AbstractBikaverin is a fungal red pigment that presents antimicrobial and antitumor activities. Therefore, this substance could be used as an alternative additive in the food and pharmaceutical industries. The aim of this work was to use response surface methodology to optimize the fermentation conditions and maximize the production of bikaverin in shake flasks. The variables investigated were agitation speed (71–289 rpm), temperature (21–35 °C), and substrate (rice) concentration in the culture medium (16.4–83.6 g/L). The agitation speed had a positive effect on red pigment production, while substrate concentration and temperature had the opposite effect. Maximum bikaverin production was predicted to occur using 289 rpm, 24.3 °C, and 16.4 g/L rice concentration. Experimental validation using 289 rpm, 28 °C, and 20 g/L rice concentration was 6.2% higher than predicted by the model. The present investigation was important for defining the best conditions for the production of bikaverin.