A central concept for understanding the mechanisms linking diversity and primary production or more general, ecosystem functioning, is resource use efficiency (RUE). It quantifies the amount of biomass production over time relative to unit resource supplied, that is, represents a quota of matter use efficiency. Given anthropogenic alterations of biogeochemical cycles, the consequent changes in supply rate and especially supply ratio of nutrients will change. Using four species of freshwater phytoplankton, and their mixture, we asked how the RUE for nitrogen and phosphorus depends on the stoichiometry of resource supply and how this differs between single species and their mixture. We conducted a factorial laboratory experiment spanning 25 different nutrient supply treatments with differing absolute and relative nitrogen (N) and phosphorus (P) concentrations. N and P supply increased biomass production and decreased C : nutrient ratios and RUE for the respective nutrient, but always significantly affected by the supply of the respective other nutrient. Biomass peaked at molar N : P supply ratios above the Redfield ratio (18–22). Species tended to respond similarly to the resource gradients. Consequently, mixtures outperformed the component species only during early growth responses, but not regarding maximum biomass and RUE. Bioassays performed at the end of the main experiment revealed predominance of N-limitation, but again strongly depending on the interaction between both nutrient gradients. Our study suggests that stoichiometric constraints of resource incorporation and RUE need to be accounted for when studying the response of phytoplankton to natural and anthropogenic variation in resource availability.