Biological nitrogen assimilation is an emerging technique for the removal and upcycling of nitrogen from wastewater in the form of microbial protein (MP). For this purpose, a mixed hydrogen-oxidizing bacteria (HOB) culture capable of growing mixotrophically was evaluated for the treatment of synthetic and real wastewater under different carbon-to-nitrogen (C/N) ratios. The same mixed HOB culture was grown under heterotrophic conditions to compare treatment and process performances in terms of ammonium nitrogen (N-NH4+) removal and assimilation, chemical oxygen demand (COD) removal, CO2 release, biomass concentration, yield and protein content. Under mixotrophic conditions, the highest biomass concentration was 342.5 mg VSS∙L−1, doubling that obtained under heterotrophic conditions (161.2 mg VSS∙L−1). Discharge limits for both COD and total N were met under mixotrophic conditions, with nitrogen removal and assimilation into protein-rich biomass achieving up to 99 %. On the contrary, under heterotrophic conditions, the high content of residual nitrogen as nitrite (up to 26 mg∙L−1 of N-NO2−) did not allow to meet discharge limits for total N. The mixed HOB culture gave promising results in terms of biomass yield (0.32 g VSS·g CODH2+acetate−1) and protein content (up to 56 % of VSS) when grown mixotrophically. Under heterotrophic conditions, instead, the biomass yield (0.25 g VSS·g CODacetate−1) and protein content (35 %) were substantially lower. This study suggests that the H2-driven assimilatory mixotrophic metabolism can be successfully applied for wastewater treatment to produce effluents that meet discharge limits while mitigating greenhouse gas emissions (CO2 and N2O) and upcycling nitrogen into MP.