The Qinghai-Tibet Plateau, known as “the third pole” of the Earth, boasts unique climatic conditions with abundant sunlight, low temperature, and significant annual temperature variations, nurturing a diverse array of soil microorganisms with rich metabolic products. In this study, 132 fungal isolates were isolated and identified from the soil samples collected in Nyingchi, Tibet, belonging to 32 genera and 59 species, while Ascomycota, Mucoromycota, and Basidiomycota accounted for 91.7%, 7.5%, and 0.8%, respectively. The evolutionary relationships among 59 representative strains were revealed by constructing a phylogenetic tree, while Penicillium, Fusarium, and Aspergillus were the most widespread fungal genera here, and the antibacterial activity of these strains was evaluated by the agar diffusion assay, leading to 27 active strains. Notably, six of them demonstrated significant activities against two or more tested bacteria. The antibacterial efficacy of the extracts of these six fungi, which were derived from four distinct media, was further evaluated at a concentration of 500 μg/mL. This study provides a valuable supplement to the investigations of cultivable soil fungi in Nyingchi, Tibet, laying a foundation for the development of soil fungi and antibacterial lead compounds in the area.