Fungi can play beneficial and detrimental roles in meat products; however, the diversity and significance of fungi in meat products are poorly understood. This study aimed to isolate and characterize fungal species from frozen beef samples collected from retail stores in the Qena Governorate, Egypt. A total of 70 frozen beef samples were collected from retail stores in Qena, Egypt. All samples were subjected to mycological examination. Fungal colonies were identified using conventional approaches, as well as the VITEK 2 system and DNA sequencing of the internal transcribed spacer region. Analyses of enzymatic activity, biofilm formation ability, and the antimicrobial resistance profiles of the isolated yeasts were also conducted. Molds and yeasts were isolated from 40% and 60% of meat samples, respectively. Mold isolates were dominated by , , and spp., whereas yeast isolates were identified as , , , , and . Compared to other yeast species, the highest production of lipase and protease was observed in species. The strongest ability to form biofilms was observed in spp., followed by . , , and . The results of antimicrobial susceptibility testing revealed that all yeast isolates showed notable resistance to fluconazole and itraconazole. A significant correlation between antimicrobial resistance and biofilm formation was observed in several species. This study highlights the importance of the dangers of yeasts in food products and the extent of their impact on public health.