Probiotic microorganisms (Saccharomyces cerevisiae var. boulardii, S. cerevisiae UFMG 905, and Lactobacillus delbrueckii UFV H2b20) were evaluated as biological control agents to reduce aflatoxin and spore production by Aspergillus parasiticus IMI 242695 in peanut. Suspensions containing the probiotics alone or in combinations were tested by sprinkling on the grains followed by incubation for seven days at 25 ∘ C. All probiotic microorganisms, in live and inactivated forms, significantly reduced A. parasiticus sporulation, but the best results were obtained with live cells. The presence of probiotics also altered the color of A. parasiticus colonies but not the spore morphology. Reduction in aflatoxin production of 72.8 and 65.8% was observed for S. boulardii and S. cerevisiae, respectively, when inoculated alone. When inoculated in pairs, all probiotic combinations reduced significantly aflatoxin production, and the best reduction was obtained with S. boulardii plus L. delbrueckii (96.1%) followed by S. boulardii plus S. cerevisiae and L. delbrueckii plus S. cerevisiae (71.1 and 66.7%, resp.). All probiotics remained viable in high numbers on the grains even after 300 days. The results of the present study suggest a different use of probiotics as an alternative treatment to prevent aflatoxin production in peanut grains.