To investigate the effect of cultural condition on the aflatoxin production of Aspergillus flayus ATCC 15517, mixed culture with Aspergillus niger, better kind of media and size of Cultural vessels were examined. YES medium was better than SLS medium for this study. Small scale test tube culture was showed the possibility to simply examine the growth, total acidity, pH and aflatoxin production during cultivation, and also could reduce the second contamination of aflatoxin B1 from large scale broth cultured. Especially ELISA method is simple, sensitive and specific and therefore well suited to small scale of test tube culture. Mixed culture significantly reduced the aflatoxin production of Aspergillus fiavus ATCC 15517 and showed almost 95% inhibition of that level during the incubtation.