The growth of the polyextremophile red microalga Galdieria sulphuraria ACUF 064 was evaluated under mixotrophy and heterotrophy in a 13-L lab-scale stirred photobioreactor, using buttermilk as a carbon source. Beforehand, G. sulphuraria ACUF 064 growth on glucose, galactose and lactose was evaluated. No significant differences were observed in terms of growth when lactose, glucose or galactose was used as a carbon source. Overall, the biomass yield on carbon was 70% higher in mixotrophy than heterotrophy for galactose (2.0 gx gC−1) and lactose (2.0 gx gC−1), while it was similar for glucose (1.5 gx gC−1). At the same time, the yield of biomass on nitrogen for cultures grown in lactose was the lowest in comparison to all the other tested substrates. This was also supported by a higher C-phycocyanin content, with 5.9% wC-PC/wx as the highest value in mixotrophy. A preliminary experiment in flasks under mixotrophic conditions with different buttermilk dilutions revealed that a dilution ratio of 40% v/v of buttermilk (corresponding to 2.0 gC L−1) was optimal for biomass production. Finally, G. sulphuraria ACUF 064 was grown in the optimal buttermilk dilution ratio in a 13 L photobioreactor. The highest biomass productivity was also obtained in mixotrophy (0.55 gx L−1 d−1), corresponding to a carbon removal of 61%. Overall, lactose-containing substrates such as buttermilk hold promise as a substrate for the growth of G. sulphuraria while revalorizing an industrial effluent.