Traditional methods for the production of food grade pigments from the fungus Monascus spp. mostly rely on submerged fermentation. However, the cell-bound nature and intracellular accumulation of pigments in Monascus spp. is a major hurdle in pigment production by submerged fermentation. The present study focused on the investigation of the effect of the antifungal agent fluconazole on red pigment production from Monascus purpureus (NMCC-PF01). At the optimized concentration of fluconazole (30 μg ml−1), pigment production was found to be enhanced by 88% after 96 h and it remained constant even after further incubation up to 168 h. Ergosterol, a sterol specific to fungi, was also extracted and estimated as a function of fungal growth. The concentration of ergosterol in fluconazole-treated fermentation broth was reduced by 49% as compared to control broth. Thus it could be responsible for facilitating the release of intracellular and cell-bound pigments. Nevertheless, the role of cell transporters in transporting out the red pigments cannot be ignored and deserves further attention. Qualitative analysis of red pigment by thin layer chromatography, UV spectroscopy and mass spectrometric analysis (ESIMS) has confirmed the presence of the well-known pigment rubropunctamine. In addition, this fermentation process produces citrinin-free pigments. This novel approach will be useful to facilitate increased pigment production by the release of intracellular or cell-bound Monascus pigments.