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Background  The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate in  Aspergillus flavus , and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time.    Results  Two field isolates of  A. flavus , AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15 mM H 2 O 2 , and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which encompassed 9 biological super-pathways and 47 sub-pathways. These metabolites were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points.    Conclusions  The moderately high aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H 2 O 2 -stressed  A. flavus  and drought-stressed hosts also suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.    Electronic supplementary material  The online version of this article (10.1186/s12866-019-1580-x) contains supplementary material, which is available to authorized users.

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time