This study investigates the potential of Pythium oligandrum (strains M1 and 00X48) as a biocontrol agent in suppressing the growth of Fusarium culmorum and the production of mycotoxins during the malting of naturally contaminated barley (Hordeum vulgare). The effects of the biocontrol agent on F. culmorum-infected barley malt (BM) were evaluated through real-time PCR and its impact on mycotoxin production was determined by quantitative analysis of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G). The effect of treatment on BM and beer quality were also determined through European Brewery Convention (EBC) standard methods. Optimal treatment with P. oligandrum strains M1 and 00X48 yielded a 59% and 48% reduction in F. culmorum contamination, by 37% and 17% lower DON, and 27% and 32% lower D3G, respectively. BM treated with both P. oligandrum strains exhibited quality enhancement; beer produced from the BM treated with P. oligandrum strain M1 resulted in no quality deterioration and with 26% and 18% less DON and D3G, respectively, transferred to the final product.