Antarctic fungi can effectively adapt to extreme environments, which leads to the production of unique bioactive compounds. Studies on the discovery of fungi in the diverse environments of Antarctica and their potential applications are increasing, yet remain limited. In this study, fungi were isolated from various substrates on the Fildes Peninsula in Antarctica and screened for their antibiosis activity against two significant plant pathogenic fungi, Botrytis cinerea and Fusarium culmorum. Phylogenetic analysis using multiple genetic markers revealed that the isolated Antarctic fungal strains are diverse, some of which are novel, emphasizing the underexplored biodiversity of Antarctic fungi. These findings suggest that these fungi have potential for the development of new antifungal agents that can be applied in agriculture to manage fungal plant pathogens. Furthermore, the antibiosis activities of the isolated Antarctic fungi were evaluated using a dual-culture assay. The results indicated that several strains from the genera Cyathicula, Penicillium, and Pseudeurotium significantly inhibited pathogen growth, with Penicillium pancosmium showing the highest inhibitory activity against Botrytis cinerea. Similarly, Aspergillus and Tolypocladium strains exhibited strong antagonistic effects against Fusarium culmorum. This study enhances our understanding of Antarctic fungal diversity and highlights its potential for biotechnological applications.