This study investigated the physicochemical properties, microbiological analysis, antifungal activity, and mycotoxin analysis of bread. Eight breads were prepared, including control Bread (CB), control Bread with calcium propionate (CPB), commercial sourdough breads (CCB), control bread made with oven-dried sourdough (SB), and bread baked with oven-dried sourdough fermented by Pediococcus pentosaceus TI6 (SB-TI6). These breads were contaminated with Aspergillus flavus and Penicillium verrucosum, and their shelf life was monitored over 7 days. The results indicated that incorporating sourdough powder into bread significantly extends the shelf life and reduces fungal contamination. This reduction resulted in lower levels of mycotoxins, with the use of dry sourdough fermented by P. pentosaceus TI6 leading to the lowest levels of aflatoxins. In the case of SB-TI6, minor levels of aflatoxin B1 (AFB1), B2 (AFB2), and G1(AFG1) were detected, with only 0.003 mg of AFG2/Kg of the product. Conversely, CB loaves exhibited the highest mycotoxin contamination levels, reaching values of 330 mg of AFB1/Kg. The findings suggested that incorporating dry sourdough in breadmaking served as a viable alternative to synthetic preservatives, effectively reducing mycotoxin levels and enhancing bread safety and shelf life.