Aflatoxins are toxic metabolites mostly produced by two fungal species (Aspergillus flavus and Aspergillus parasiticus) in cereal grains, groundnuts, corn and rice. In the present investigation the HPLC technique was used for estimation of aflatoxins produced by each Aspergillus species either without or with co-cultivation of B. subtilis. The antitoxin production activity of Bacillus subtilis was detected when being co-cultivated with Aspergillus flavus and Aspergillus parasiticus under different cultural conditions ( pH, temperatures , incubation periods and two types of media). The thermal stability of the antitoxin production element by bacterium was post-heating to 80, 100 ºC and autoclaving at 121 ºC. Maximum antiaflatoxigenic effect of this bacterium was observed at 25 ºC and pH 10 after four days cultivation with Aspergillus flavus, while, the activity appeared at 25 ºC and pH 10 after ten days when cultivated with Aspergillus parasiticus. The antitoxin production metabolites are shown to be thermostable at 80, 100ºC and autoclaving at 121ºC for 30 minutes.