The microalga Chlorella vulgaris can produce carbohydrates and lipids within short periods with high lipid productivity. Biofilm reactors can be used for microalga cultivation; however, the research on the use of biofilm reactors as a platform for algal biomass and oil production under mixotrophic conditions is limited. The goals of our work were 1) to develop a mixotrophic algal biofilm reactor using glycerol and urea as carbon and nitrogen sources; 2) to quantify biomass, total lipid, glycerol and urea consumption rates and estimate fatty acid profiles; and 3) to measure dissolved oxygen and pH depth profiles using microelectrodes under dark and light conditions. A membrane bioreactor in continuous mode with recycle under 50 μmol photons m− 2 s− 1 light irradiance was used to grow the algae. No exogenous CO 2 was supplied. The productivities were 9.27 ± 0.47 g DW m− 2 d− 1 and 12.64 ± 0.94 g DW m− 2 d− 1 when 2 and 5 g L− 1 initial glycerol concentrations were used. The total lipid contents were 13 ± 0.02% and 23.91 ± 0.03%, respectively. Depth profile measurements showed a maximum dissolved oxygen concentration of 3.5 mg L− 1 near the surface and a minimum of 1.4 mg L− 1 at the bottom of the biofilm. The pH in the biofilms did not vary with the depth, and it was around 6.8 in the dark and 7.5 in the light. The main fatty acids produced were C18:1, C18:2 and C16:0, and there was a low saturated fatty acid content of 23.37%. The designed biofilm reactor allowed C. vulgaris growth using very low light with a lower water demand. The composition of fatty acids detected in Chlorella vulgaris oil was significantly polyunsaturated. The biofilm growth mode affected C. vulgaris metabolism such that the cell met its CO 2 requirements internally and a high oil yield was obtained without nitrogen starvation.