Aspergillus fumigatus and Rhizopus microsporus are globally distributed pathogenic fungi responsible for causing a wide spectrum of infections called aspergillosis and mucormycosis, respectively. The diagnosis of these infections still faces challenges due to various factors such as non-specific clinical symptoms, overlapping risk factors, non-discrimination between colonization and invasive infection and limited sensitivity to already existing diagnostic tools. Moreover, the increase in antifungal resistance and vaccine unavailability to these infections emphasizes the critical need for specific and timely diagnostics. One area of research is using mycotoxins and microbial metallophores, specifically siderophores, which play crucial roles in fungal growth, survival, and pathogenesis; as a result, their complex structures could enable high diagnostic specificity. Liquid chromatography-mass spectrometry- based metabolomics can provide accurate and rapid detection of these fungal metabolites and can also specify a comprehensive overview of pathogen-induced changes to the host cells following infection. Therefore, the main goal of this thesis is the liquid chromatography- mass spectrometry (MS) based characterization of A. fumigatus and R. microsporus siderophores and other metabolites. The thesis focuses on the following four topics: introduction of infection metallomics, characterization of R. microsporus siderophores, characterization of A. fumigatus siderophores and mycotoxins to distinguish between colonization and invasion, as well as during inter-kingdom interaction with bacterial pathogen-Pseudomonas aeruginosa, host cells-neutrophils and polymycovirus and finally, infection metallomics based diagnosis of human and equine aspergillosis. The theoretical part covers general information on A. fumigatus and R. microsporus, related infections, secondary metabolites, and specific knowledge about each mentioned topic. Experimental, results and discussion parts are divided into corresponding sections to each topic. The first section introduces infection metallomics, a MS platform based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. The second section of this thesis includes R. microsporus in vitro cultivation, followed by liquid chromatography- mass spectrometry analysis of its siderophores. The third section involves in vitro cultivation of A. fumigatus with subsequent growth stage-specific siderophore and mycotoxin quantification to distinguish between colonization and invasive infection. Additionally, this section deals with the time course quantification of A. fumigatus secondary metabolites during its interaction with neutrophils, bacterial pathogen P. aeruginosa and polymycovirus. The analysis is performed using developed metabolite extraction protocol and mass spectrometry methods. The fourth and last part focuses on applying infection metallomics-based diagnosis in human invasive pulmonary aspergillosis and equine aspergillosis. Taken together, this thesis shows the performance of the non-invasive infection metallomics armoury and makes difference towards the standard serology, cultivation, microscopy, or nucleic acid analyses routinely used in invasive fungal infection diagnostics. The thesis documents why the innovative non-invasive approach based on mass spectrometry based microbial metallophores detection with benefit of isotope data filtration is inherently more sensitive and specific in selected applications than classical clinical standards.Aspergillus fumigatus and Rhizopus microsporus are globally distributed pathogenic fungi responsible for causing a wide spectrum of infections called aspergillosis and mucormycosis, respectively. The diagnosis of these infections still faces challenges due to various factors such as non-specific clinical symptoms, overlapping risk factors, non-discrimination between colonization and invasive infection and limited sensitivity to already existing diagnostic tools. Moreover, the increase in antifungal resistance and vaccine unavailability to these infections emphasizes the critical need for specific and timely diagnostics. One area of research is using mycotoxins and microbial metallophores, specifically siderophores, which play crucial roles in fungal growth, survival, and pathogenesis; as a result, their complex structures could enable high diagnostic specificity. Liquid chromatography-mass spectrometry- based metabolomics can provide accurate and rapid detection of these fungal metabolites and can also specify a comprehensive overview of pathogen-induced changes to the host cells following infection. Therefore, the main goal of this thesis is the liquid chromatography- mass spectrometry (MS) based characterization of A. fumigatus and R. microsporus siderophores and other metabolites. The thesis focuses on the following four topics: introduction of infection metallomics, characterization of R. microsporus siderophores, characterization of A. fumigatus siderophores and mycotoxins to distinguish between colonization and invasion, as well as during inter-kingdom interaction with bacterial pathogen-Pseudomonas aeruginosa, host cells-neutrophils and polymycovirus and finally, infection metallomics based diagnosis of human and equine aspergillosis. The theoretical part covers general information on A. fumigatus and R. microsporus, related infections, secondary metabolites, and specific knowledge about each mentioned topic. Experimental, results and discussion parts are divided into corresponding sections to each topic. The first section introduces infection metallomics, a MS platform based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. The second section of this thesis includes R. microsporus in vitro cultivation, followed by liquid chromatography- mass spectrometry analysis of its siderophores. The third section involves in vitro cultivation of A. fumigatus with subsequent growth stage-specific siderophore and mycotoxin quantification to distinguish between colonization and invasive infection. Additionally, this section deals with the time course quantification of A. fumigatus secondary metabolites during its interaction with neutrophils, bacterial pathogen P. aeruginosa and polymycovirus. The analysis is performed using developed metabolite extraction protocol and mass spectrometry methods. The fourth and last part focuses on applying infection metallomics-based diagnosis in human invasive pulmonary aspergillosis and equine aspergillosis. Taken together, this thesis shows the performance of the non-invasive infection metallomics armoury and makes difference towards the standard serology, cultivation, microscopy, or nucleic acid analyses routinely used in invasive fungal infection diagnostics. The thesis documents why the innovative non-invasive approach based on mass spectrometry based microbial metallophores detection with benefit of isotope data filtration is inherently more sensitive and specific in selected applications than classical clinical standards.