Abstract The influence of nitrate supplementation and ultraviolet-B (UVB; 280–320 nm) enhancement was tested on a coastal phytoplankton assemblage from eastern Canada exposed to ambient or supplemental UVB irradiance, equivalent to a local 60% ozone depletion. During a 10 d-long mesocosm experiment, 24-h surface bag incubations were repeated three times with half the bags supplemented with nitrate, phosphate and silicate while the other half received only phosphate and silicate. At beginning and the end of these 24-h surface incubations, chlorophyll fluorescence measurements were performed and the abundance of photoprotective pigments diadinoxanthin and diatoxanhtin, of the PSII reaction center D1 protein and of the large subunit of the ribulose-1,5-biphosphate carboxylase/oxygenase enzyme (RuBisCO LSU) was determined. Results showed that as long as nutrients were abundant inside the mesocosms, phytoplankton exposed to supplemental UVB at the surface were able to maintain their maximal quantum yield of PSII fluorescence. As nutrients became limiting inside the mesosocoms, however, phytoplankton showed an increased sensitivity to supplemental UVB and suffered more net photodamage to the PSII reaction centers (seen as decreases in D1 protein abundance). Supplemental UVB also resulted in low abundance of RuBisCO LSU and exacerbated photoinhibition compared to the phytoplankton exposed to surface ambient irradiance. Supplementing nitrate during this nutrient deficient period limited the inactivation of the PSII reaction centers and lowered photoinhibition. Nitrate supplementation had no clear effect on the abundance of the D1 protein but it helped the community to maintain a greater abundance of RuBisCO LSU. Overall, results from this study suggest that the sensitivity of the RuBisCO enzyme to the combined effects of supplemental UVB and nitrate limitation can influence the tolerance of PSII to UVB stress.