ATP-binding cassette (ABC) transporters mediate active efflux of natural and synthetic toxicants and are considered to be important for drug tolerance in microorganisms. In biological control agents (BCA), ABC transporters can play important roles in antagonism by providing protection against toxins derived from the fungal prey and by mediating the secretion of endogenous toxins. In the present study, we generated deletion and complementation strains of the ABC transporter abcG5 in the fungal BCA Clonostachys rosea to study its role in xenobiotic tolerance and antagonism. Gene expression analysis shows induced expression of abcG5 in the presence of the Fusarium mycotoxin zearalenone (ZEA), secreted metabolites of F. graminearum, and different classes of fungicides. Phenotypic analysis of abcG5 deletion and complementation strains showed that the deletion strains were more sensitive towards F. graminearum culture filtrates, ZEA, and iprodione- and mefenoxam-based fungicides, thus suggesting the involvement of abcG5 in cell protection. The ΔabcG5 strains displayed reduced antagonism towards F. graminearum in a plate confrontation assay. Furthermore, the ΔabcG5 strains failed to protect barley seedlings from F. graminearium foot rot disease. These data show that the abcG5 ABC transporter is important for xenobiotic tolerance and biocontrol traits in C. rosea.