Biological control is a promising approach to reduce plant diseases caused by nematodes to ensure high productivity in agricultural production. Large-scale analyses of genetic variation in fungal species used for biocontrol can generate knowledge regarding interaction mechanisms that can improve efficacy of biocontrol applications. In this study, we performed a genome-wide association study (GWAS) for in vitro antagonism against the root lesion nematode Pratylenchus penetrans in 53 previously genome re-sequenced strains of the biocontrol fungus Clonostachys rosea. Nematode mortality in C. rosea potato dextrose broth (PDB) culture filtrates was highly variable and showed continuous variation (p < .001) between strains, indicating a polygenic inheritance. Twenty-one strains produced culture filtrates with higher (p ≤ .05) nematode mortality compared with the PDB control treatment, while ten strains lowered (p ≤ .05) the mortality. The difference in in vitro antagonism against P. penetrans correlated with antagonism against the soybean cyst nematode Heterodera glycines, indicating lack of host specificity in C. rosea. An empirical Bayesian multiple hypothesis testing approach identified 279 single nucleotide polymorphism markers significantly (local false sign rate < 10(-10)) associated with the trait. Genes present in the genomic regions associated with nematicidal activity included several membrane transporters, a chitinase and genes encoding proteins predicted to biosynthesize secondary metabolites. Gene deletion strains of the predicted nonribosomal peptide synthetase genes nps4 and nps5 were generated and showed increased (p ≤ .001) fungal growth and conidiation rates compared to the wild type. Deletion strains also exhibited reduced (p < .001) nematicidal activity and reduced (p ≤ .05) biocontrol efficacy against nematode root disease and against fusarium foot rot on wheat. In summary, we show that the GWAS approach can be used to identify biocontrol factors in C. rosea, specifically the putative nonribosomal peptide synthetases NPS4 and NPS5.