Biological control fungi play an important role in the management of plant-parasitic nematodes; however, the molecular basis underlying their diverse biocontrol strategies remains incompletely understood. In this study, a comparative genomic analysis was performed on four representative biocontrol fungi, Purpureocillium lilacinum PLFJ-1, Trichoderma harzianum CBS 226.95, Pochonia chlamydosporia 170, and Aspergillus niger CBS 513.88. Genome comparison revealed substantial variation in genome size, GC content, and gene composition among the four fungi. Phylogenetic analysis divided these fungi into two major clades corresponding to distinct evolutionary lineages. Orthogroup analysis identified both a conserved core gene set and species-specific gene repertoires. Functional annotation based on KEGG, KOG, and GO indicated a high degree of conservation in core metabolic processes, catalytic activities, and cellular components, accompanied by distinct differences in specific functional categories. Further comparative analyses demonstrated pronounced variation in the composition and abundance of carbohydrate-active enzymes (CAZymes) and peptidases, as well as a notable expansion and enrichment of S8 subtilisin-like serine peptidases in the nematode-parasitic fungi P. lilacinum and P. chlamydosporia. Secondary metabolite analysis identified diverse biosynthetic gene clusters, with differences in PKS and NRPS distribution among species. Together, these findings suggest that distinct enzymatic and metabolic gene repertoires, particularly expansions of S8 serine peptidases and specific CAZyme families, may contribute to the biocontrol potential of these fungi.