Aflatoxin production by isolates ofAspergillus flavus varies, ranging from highly toxigenic to completely atoxigenic. Several mechanisms have been identified which regulate aflatoxin production including medium carbon source and oxidative stress. In recent studies, aflatoxin production has been implicated in partially ameliorating oxidative stress inA. flavus. To better understand the role of aflatoxin production in oxidative stress responses, a selection of toxigenic and atoxigenic isolates ofA. flavus with moderate to high oxidative stress tolerance were exposed to increasing concentrations of H2O2 in both aflatoxin-conducive and non-conducive media. Mycelial mats were collected for global transcriptome sequencing followed by differential expression, functional prediction, and weighted co-expression analyses. Oxidative stress and medium carbon source had a significant effect on the expression of several secondary metabolite gene clusters including those for aflatoxin, aflatrem, aflavarin, cyclopiazonic acid, and kojic acid. Atoxigenic biological control isolates showed less differential expression under stress than other atoxigenic isolates suggesting expression profiles may be useful in screening. Increasing stress also resulted in regulation of SakA/Hog1 and MpkA MAP kinase signalling pathways pointing to their potential roles in regulating oxidative stress responses. Their expression was also influenced by medium carbon source. These results suggest that aflatoxin production along with that of other mycotoxins may occur as part of a concerted coping mechanism for oxidative stress and its effects in the environment. This mechanism is also regulated by availability of simple sugars and glycolytic compounds for their biosynthesis.