The application of fungicides is so critical, especially in greenhouses, to avoid fungal infections. Carbendazim, an inhibitor of tubulin biosynthesis, is the most widely known broad-spectrum benzimidazole fungicide. The application of carbendazim affects other beneficial fungi as well. Paecilomyces lilacinus 36-1 (Pl36-1) is a beneficial fungus used for biological control, and the most effective biocontrol agents of nematode eggs. The Pl36-1 is sensitive to carbendazim (0.3 μg/ml). There is a general consensus that the mechanisms of resistance to carbendazim in the β-tubulin gene have been analyzed in detail. However, no studies were conducted on P. lilacinus strains. In the present study, two carbendazim-resistant mutants of Pl36-1, P50 and P100, were obtained from UV exposure and tested. The β-tubulin gene fragments were cloned and sequenced in the three strains, Pl36-1, P50 and P100. The resistance to carbendazim was developed when amino acid substitutions occurred at β-tubulin gene positions of S145A, T185A and F200Y. The β-tubulin gene was overexpressed in Pl36-1 strains. The β-tubulin expression level of the overexpressed mutant (PL3), quantified by qRT-PCR, was increased 4-folds over its normal level in Pl36-1. In vitro, the PL3 was resistant to carbendazim with maintaining growth, sporulation and pathogenicity rates. Three-year field trial demonstrated that P100 and PL3 strains exhibited carbendazim resistance combined with high nematode reduction and yield improvement.