Aspergillus flavus is a saprophytic fungus which can grow on corn and produce aflatoxins which render it unsafe for consumption as food and feed. In this study, aflatoxin and non-aflatoxin producing isolates of A. flavus were grown separately on wet (20% water added), sterile or non-sterile cracked corn. Wet and dry cracked corn controls were included as needed. Secondary metabolic volatiles were identified and aflatoxin concentrations determined over a 12-day period. Volatiles unique to the toxigenic A. flavus isolates were determined by comparison with volatiles produced by the respective corn controls and the non-toxigenic A. flavus isolate. The number and identity of the volatiles produced by these A. flavus isolates varied by isolate, whether sterile or non-sterile corn was the substrate, and the sampling day. Overall, most of the volatiles were produced before day 8 after inoculation. Aflatoxin production was 10-fold lower on the sterile corn, compared to the non-sterile corn. Volatiles unique to the aflatoxin producing isolates were identified on both substrates after comparison with those produced by the non-aflatoxin producing isolate, as well as the corn control samples. Results indicate that several factors (substrate, fungal isolate, culture age) affect volatile and aflatoxin production by A. flavus.