Microalgae are known for producing various biotechnological products. Moreover, they absorb nutrients from dairy wastewater, grow well, and accumulate valuable compounds faster. In this study, photoautotrophic and mixotrophic cultivation with different initial lactose concentrations present in cheese whey (CW) were established to investigate their effect on cell concentration (Xm, mg L-1), cell productivity (Px, mg L-1day-1), and specific cell growth (µmax, day-1) of Chlorella vulgaris, Dunaliella tertiolecta, and Tetradesmus obliquus. The biomass production of C. vulgaris (Xm= 1,520 ± 30.3 mg L-1, Px = 147 ± 3.00 mg L-1, and µmax = 0.150 ± 0.00 mg L-1) in mixotrophic culture with 10.0 g L-1 of lactose, the main constituent of CW, was notably enhanced by 55% in comparison with their photoautotrophic cultures, whereas a lower effect of these lactose concentrations on cell growth was observed in T. obliquus and D. tertiolecta. Thus, mixotrophic cultivation of C. vulgaris using CW as a carbon and energy source could be considered a feasible alternative to obtain high value-added biomass. Microalgae are known for producing various biotechnological products. Moreover, they absorb nutrients from dairy wastewater, grow well, and accumulate valuable compounds faster. In this study, photoautotrophic and mixotrophic cultivation with different initial lactose concentrations present in cheese whey (CW) were established to investigate their effect on cell concentration (Xm, mg L-1), cell productivity (Px, mg L-1day-1), and specific cell growth (µmax, day-1) of Chlorella vulgaris, Dunaliella tertiolecta, and Tetradesmus obliquus. The biomass production of C. vulgaris (Xm= 1,520 ± 30.3 mg L-1, Px = 147 ± 3.00 mg L-1, and µmax = 0.150 ± 0.00 mg L-1) in mixotrophic culture with 10.0 g L-1 of lactose, the main constituent of CW, was notably enhanced by 55% in comparison with their photoautotrophic cultures, whereas a lower effect of these lactose concentrations on cell growth was observed in T. obliquus and D. tertiolecta. Thus, mixotrophic cultivation of C. vulgaris using CW as a carbon and energy source could be considered a feasible alternative to obtain high value-added biomass.