The efficacy of calcium propionate at 2 different doses (0.5% and 1%) against growth and aflatoxins production by Aspergillus flavus (A-2092) was investigated in vitro on Czapek yeast extract agar at different levels of water activity (a(w)) in the range of 0.94 to 0.996a(w). A. flavus spores germinated on all calcium propionate and a(w) treatments; however, 1% calcium propionate at 0.94 a(w) delayed the germination process for up to 10 d. The growing rate of mycelia was slower (0.28 mm/d) at 1% calcium propionate and 0.94 a(w). Aflatoxins (B1, B2, G1, and G2) were also produced minimally (36.1, 1, 1.86, and 1.01 ng/g of media, respectively) at the aforementioned dose rate of calcium propionate and water activity. It was concluded that addition of calcium propionate and a(w) amelioration can prove effective tools for suppressing the germination, growth rate, and aflatoxins production by A. flavus in substrate.