We used ⁶⁰Co-γ ray and electron beam irradiation to mutate Bacillus velezensis to breed genetically stable mutant strains. The mutant strains were screened and re-screened by plate confrontation and the four-point method to study their antibacterial effects on Colletotrichum gloeosporioides. The results demonstrated that the D₁₀ value of Bacillus velezensis suspension irradiated by ⁶⁰Co-γ ray was 2 366 Gy, and the lethality rate increased with an increase in the irradiation coefficient dose in the range of 100-2 000 Gy. The lethality rate of Bacillus velezensis was 81.8% at 2 000 Gy irradiation coefficient dose, and five mutants (B004, B112, B114, B117, and B118) were screened. The inhibition rate against Colletotrichum gloeosporioides increased from 51.2% of the original strain to 57.5%, 58.0%, 57.0%, 57.7%, and 59.9%, respectively. The Bacillus velezensis suspension was irradiated by an electron beam (10 MeV), the D₁₀ value was 499 Gy, and the lethality rate increased with an increase in the irradiation coefficient dose in the range of 100-600 Gy. The lethality rate of Bacillus velezensis was 91.8% at 400 Gy irradiation coefficient dose. Two mutant strains (D115 and D243) were screened, and their inhibition rates against Colletotrichum gloeosporioides increased from 55.2% of the emitted strains to 58.4 and 58.1%, respectively. The inhibitory effects of B004, B112, B117, and B118 mutants on Colletotrichum gloeosporioides were genetically stable within six generations. In conclusion, γ-ray and electron beam irradiation have potential application value in the mutation breeding of Bacillus velezensis. The results provide a basis for Bacillus velezensis mutation breeding and a theoretical reference for the biological control of Colletotrichum gloeosporioides.