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Abstract The Antarctic continent is one of the most inhospitable places on earth, where living creatures, mostly represented by microorganisms, have specific physiological characteristics that allow them to adapt to the extreme environmental conditions. These physiological adaptations can result in the production of unique secondary metabolites with potential biotechnological applications. The current study presents a genetic and antibacterial characterization of four Antarctic fungi isolated from soil samples collected in Pedro Vicente Maldonado Scientific Station, at Fort William Point, Greenwich Island, Antarctica. Based on the sequences of the internal transcribed spacer (ITS) region, the fungi were identified as Antarctomyces sp., Thelebolus sp., Penicillium sp., and Cryptococcus gilvescens. The antibacterial activity was assessed against four clinical bacterial strains: Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, and Staphylococcus aureus, by a modified bacterial growth inhibition assay on agar plates. Results showed that C. gilvescens and Penicillium sp. have potential antibiotic activity against all bacterial strains. Interestingly, Thelebolus sp. showed potential antibiotic activity only against E. coli. In contrast, Antarctomyces sp. did not show antibiotic activity against any of the bacteria tested under our experimental conditions. This study highlights the importance of conservation of Antarctica as a source of metabolites with important biomedical applications.