Microalgae biomass is a viable feedstock for a wide range of industries. Recently, there has also been interest in the ability of microalgae biomass applications for biofuel production. In the meantime, the cultivation of microalgae biomass requires high energy costs, and the application of microalgae for technical purposes is still problematic. A significant part of the cost of biomass arises from the nutrients used for cultivation. Chemical compounds included in the microalgae cultivation media can be replaced by suitable wastes containing nitrogen, phosphorus, and other elements. This could reduce the microalgae biomass cultivation price and allow cheaper biomass to be used for biofuel production. The aim of this work was to comprehensively investigate and optimize the growth process of microalgae using liquid waste (liquid waste after biogas production from sewage sludge and distillers’ grain) as a source of nitrogen and phosphorus, and technical glycerol as a carbon source. It was found that higher levels of waste in the cultivation media were found to inhibit the accumulation of microalgal biomass, with the optimum level corresponding to a nitrogen concentration of 0.08 g/L. The influence of technical glycerol from biodiesel production on the yield of microalgal biomass was investigated, and it was found that the addition of 6% glycerol allows an increase in the concentration of microalgal biomass in the cultivation media, from 18.1 to 20.6%.