The purpose of this work was to investigate the potential of two nontoxigenic Aspergillus section Nigri species (Aspergillus tubingensis and Aspergillus japonicus) to influence the in vitro ochratoxin A (OTA) production of three toxigenic Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33) from Greek vineyards of different geographical areas. OTA accumulation was evaluated by inoculation of 0:100, 25:75, 50:50, 75:25, and 100:0 ratios of mixed spore suspensions on a synthetic grape juice medium for up to 28 days at different temperatures (15, 20, and 25°C), water activity (aw) levels (0.95 and 0.98 aw) and incubation time (7, 14, 21, and 28 days). Results confirmed that environmental factors and fungal species had a significant effect on OTA production. Specifically, maximum OTA concentration for Ac-28 (3.21 μg g(-1)) and Ac-29 (7.69 μg g(-1)) was observed at 20°C/0.98 aw and for Ac-33 (9.13 μg g(-1)) at 15°C/0.95 aw, regardless of incubation time. Moreover, A. tubingensis had no significant influence on OTA concentration of all toxigenic isolates assayed, regardless of temperature, aw, and incubation time. On the other hand, the presence of A. japonicus slightly inhibited OTA production of Ac-29 and Ac-33, while for Ac-28, stimulation of OTA was observed in some cases. Overall, lower aw levels reduced OTA accumulation for Ac-28 and Ac-29, regardless of temperature, inoculum ratio, and time. On the contrary, for Ac-33, low aw increased OTA production, regardless of the investigated parameters. The importance of this study concerns the understanding of interspecific interactions on OTA diffusion by A. carbonarius in an attempt to find ways to prevent the presence of toxins in grapes and their derivatives.