Trichosporon asahiiis an opportunistic pathogen that can cause severe or even fatal infections in patients with low immune function.sPLA2plays different roles in different fungi and is also related to fungal drug resistance. However, the mechanism underlying its drug resistance to azoles has not yet been reported inT. asahii. Therefore, we investigated the drug resistance ofT. asahii PLA2(TaPLA2) by constructing overexpressing mutant strains (TaPLA2OE). TaPLA2OEwas generated by homologous recombination of the recombinant vector pEGFP-N1-TaPLA2, induced by the CMV promoter, withAgrobacterium tumefaciens. The structure of the protein was found to be typical of sPLA2, and it belongs to the phospholipase A2_3 superfamily. TaPLA2OEenhanced antifungal drug resistance by upregulating the expression of effector genes and increasing the number of arthrospores to promote biofilm formation. TaPLA2OEwas highly sensitive to sodium dodecyl sulfate and Congo red, indicating impaired cell wall integrity due to downregulation of chitin synthesis or degradation genes, which can indirectly affect fungal resistance. In conclusion,TaPLA2overexpression enhanced the resistance to azoles ofT. asahiiby enhancing drug efflux and biofilm formation and upregulating HOG-MAPK pathway genes; therefore, it has promising research prospects.