This study examines the use of Pythium oligandrum as a biological control agent to suppress Fusarium culmorum growth and mycotoxin production during the malting of wheat (Triticum aestivum L.). The optimum application phase and dose of P. oligandrum were studied. The effect of P. oligandrum on F. culmorum-infected wheat was quantitatively evaluated through RT-PCR along with deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) content in wheat malt (WM). In addition, the effect of P. oligandrum treatment on WM quality was quantified by European Brewery Convention standard methods. Treatment by P. oligandrum in the steeping stage (2 g P. oligandrum/kg wheat) yielded optimal suppression of Fusarium contamination (20%) and mycotoxin content (17% DON and 21% D3G) relative to untreated WM. Treatment dose showed a maximum effect between 0.6 and 1 g P. oligandrum/kg of wheat, resulting in 12–14% Fusarium contamination, 13–14% DON, and 15–18% D3G, relative to untreated malt. Wheat malts treated with P. oligandrum showed no deterioration of malting or brewing quality. Display omitted •Biological control of fungal contamination in malting is desirable.•Pythium oligandrum was used to combat Fusarium culmorum in infected wheat.•P. oligandrum was effective against fungal growth and mycotoxin production.•Optimum application (steeping) and dose (0.6–1 g/kg wheat) were established.•The brewing quality of wheat malt was not affected by P. oligandrum.