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¹ Kütahya Health Sciences University, Faculty of Engineering and Natural Sciences, Department of Molecular Biology and Genetics, Kütahya, Türkiye DOI : 10.38042/biotechstudies.1739583 - Fungi are rich sources of medicinal bioactive metabolites. The fungi used in this study were identified as Rhizomucor pusillus and Aspergillus niger through multilocus gene sequencing. HPLC was used to analyze the cell-free culture filtrate (CFCF) of the fungi. The most prevalent organic acid in A. niger was tartaric acid, whereas the most predominant acid in R. pusillus was succinic acid. Furthermore, chlorogenic acid was the most abundant phenolic compound in both CFCFs. The impacts of the CFCFs as antibacterial agents against Gram (+) bacteria Staphylococcus aureus, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus sp. (MRCoNS) and Gram (-) bacteria Pseudomonas aeruginosa, Escherichia coli, a yeast Candida parapsilosis and a filamentous fungus A. niger were also investigated. CFCF of A. niger was found to exhibit significant antimicrobial activity by acting on a wider range of microorganisms than CFCF of R. pusillus. Especially, CFCFs of A. niger and R. pusillus demonstrated the highest levels of activity at a concentration of 0.125 mL/mL against MRSA and C. parapsilosis, respectively. This study offers the first comparison of organic acids and phenolics in R. pusillus and A. niger CFCFs, linking their distinct antimicrobial profiles to potential use in fungal-based antimicrobial formulations. Keywords : Fungi Antibacterial activity Antifungal activity Bioactive metabolites HPLC