Abstract: The objective is to breed strains producing high-yield gliotoxin. Trichoderma virens TY-009 was used as the original strain. The method of atmospheric and room temperature plasma (ARTP) was used to induce the mutation of Trichoderma virens producing gliotoxin to obtain mutants. The time gradients were set up. The yield of gliotoxin in different time was detected. Eight positive mutants were obtained through plate screening and shaking culture selection. The highest production of gliotoxin was 0.86 mg/mL, which increased by 45.76% compared with that of the original strain. ARTP was proved to be a fast and efficient method, and had better mutation of Trichoderma virens. It was believed that ARTP had a great application potential for improving the production of gliotoxin.