Prevention in the field of mycotoxin-producing fungi is the most effective strategy for controlling the presence of mycotoxins in foods. Chemical fungicides are widely used to protect crops, so their implications on mycotoxin production need to be considered. Therefore, the aim of this study was to evaluate the effect in vitro and on grapes of five fungicides commonly used on grape cultures in Brazil on Aspergillus carbonarius growth and ochratoxin A (OTA) production. At the doses recommended by manufacturers, most fungicides significantly reduced A. carbonarius growth and OTA production in vitro, whereas this effect was influenced by the type of fungicide, dose, and temperature. Temperature was the main factor that influenced the effectiveness of fungicides. In general, at 15°C, fungicides showed the greatest reduction on fungal growth and OTA production. Fungicide effect on grapes was different to that on a semisynthetic grape medium. All fungicide doses were not effective at controlling A. carbonarius in grapes. Thus, the direct effect of fungicides on grapes must be studied to obtain a better approximation of field conditions. The results indicate that the use of fungicides at the doses recommended by manufacturers is better than the application at low doses. This study showed that at the lowest doses, where fungal growth is not inhibited, fungicides positively stimulate OTA production.