In this study, the antifungal activity of white mustard bran (MB), a by-product of mustard (Sinapis alba) milling, and white mustard seed flour (MF) was tested against mycotoxigenic fungi in the agar diffusion method. The results obtained were posteriorly confirmed in a quantitative test, determining the minimum concentration of extract that inhibits the fungal growth (MIC) and the minimum concentration with fungicidal activity (MFC). Since MF demonstrated no antifungal activity, the MB was stored under different temperature conditions and storage time to determine its antifungal stability. Finally, an in situ assay was carried out, applying the MB as a natural ingredient into the dough to avoid P. commune CECT 20767 growth and increase the bread shelf life. The results demonstrated that the antifungal activity of MB was dose-dependent. The higher assayed dose of MB (10 g/kg) reduced the fungal population in 4.20 Log CFU/g regarding the control group. Moreover, the shelf life was extended four days compared to the control, equaling its effectiveness with the synthetic preservative sodium propionate (E-281). Therefore, MB could be an alternative to chemical additives in bread formulations since it satisfies consumer requirements. Also, the formulation of bread with MB valorizes this by-product generated during mustard seed milling, thereby helping the industry move forward sustainably by reducing environmental impact.