<p>Aflatoxins (AFs) are highly toxic second metabolites produced by <i>Aspergillus flavus </i>and<i> A. parasiticus</i>. They are widely detected in cereals, spices, and drinks worldwide. Aflatoxin contamination of foods and crops poses a high health risk for humans and livestock. It is well known that environmental conditions and substrates could influence fungal growth and aflatoxin production. This study tested the effect of water activity (0.82, 0.86, 0.90, 0.94, and 0.98 a_w) and incubation temperatures (20°, 27°, and 35°C) on the growth and aflatoxin production of <i>A. flavus </i>and <i>A. parasiticus</i> on ground flax seeds and ground niger seeds. The effect of oil contents of ground niger seeds on fungal growth and aflatoxin production was also investigated in this study.</p><p> These two fungal species could not grow on any of the tested substrates with 0.82 a_w at 20°, 27°, or 35°C. <i>Aspergillus flavus</i> grew most rapidly on flax seeds with 0.90 a_w at 27°C and also 0.94 a_w at 27° or 35°C. However, on niger seeds, <i>A. flavus </i>grew best at 0.90 or 0.94 a_w incubated at 35°C as well as at 0.94 or 0.98 a_w incubated at 27°C. <i>Aspergillus parasiticus</i> showed the optimum growth on flax seeds with 0.90 a_w at 35°C, whereas on niger seeds, the optimum occurred on seeds with 0.90 a_w at 35°C and also on seeds with 0.94 a_w at 27° or 35°C. The optimum conditions for <i>A. flavus </i>to produce high levels of aflatoxins (270-299 μg/kg) on flax seeds were 0.90 a_w at 35°C; whereas, the optimum conditions for <i>A. flavus </i>to produce aflatoxin (203-278 μg/kg) on niger seeds were 0.90 or 0.98 a_w at 27°C and also 0.90 a_w at 35°C. <i>Aspergillus parasiticus</i> produced high levels of aflatoxins (284-365 μg/kg) on flax seeds under the following three conditions, 0.86 or 0.98 a_w at 35°C and 0.94 a_w at 27°C; <i>A. parasiticus</i> produced 200-384 μg/kg of aflatoxins on niger seeds under nine out of 12 tested incubation conditions.</p><p> Reducing mean oil contents from 35.2 to 10.5% of ground niger seeds had very little effect on the growth of the two fungi but significantly decreased their aflatoxin production under certain incubation conditions. On de-oiled niger seeds inoculated with <i>A. flavus</i>, only 13μg/kg of AFB1 was found on seeds with 0.94 a_w at 27°C; whereas, on de-oiled niger seeds inoculated with <i>A. parasiticus</i>, high levels of aflatoxins (245-345 μg/kg) were only detected under the three following incubation conditions, 0.90 or 0.94 a_w at 27°C, and 0.86 a_w at 35°C.</p> This study showed that the optimum growth and aflatoxin production by <i>A. flavus </i>and<i> A. parasiticus </i>were not identical and influenced by incubation conditions, including temperature, water activity, and growth substrates. The results of this study could help establish guidelines for post-harvest and storage conditions for oil seeds to prevent fungal growth and aflatoxin formation.