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Atmospheric changes are leading to the browning of northern lakes (i.e. increases in catchment-derived dissolved organic matter [DOM]), consequently altering phytoplankton biomass and community composition. We hypothesised that lake browning and the concurrent increase in nutrients drive a shift towards greater cyanobacteria biomass. We further hypothesised that, as a consequence of this shift in phytoplankton, the content of omega-3 (n-3) essential fatty acids (EFA) in seston would decline, affecting the subsequent transfer of EFA to consumers across the plant-animal interface in pelagic regions of lakes. We tested these hypotheses in the epilimnion of 30 temperate lakes in Ontario (Canada), representing a gradient of lake browning, with dissolved organic carbon (DOC) ranging from 2 to 10 mg/L and total phosphorus ranging from 6.0 to 48.5 mu g/L. In each of these lakes, the concentration and composition of DOM, the biomass of phytoplankton and cyanobacteria, and the EFA content of seston, cladocerans, and copepods were measured. An increase in aromatic DOM was associated with increased phytoplankton and cyanobacteria biomass. Due to the lower content of the EFA eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in cyanobacteria, this increase in phytoplankton biomass was associated with a decline in EPA and DHA content in lake seston. However, there was no significant change in EFA content of cladocerans and copepods. This homeostatic (diet-independent) EFA composition in zooplankton suggested that, as the phytoplankton community shifted towards more cyanobacteria with lower EFA content, the cladocerans and copepods may have met their nutritional requirements by relying on alternative food sources (e.g. heterotrophic ciliates and flagellates) capable of either trophically upgrading phytoplankton-produced EPA and DHA, or synthesising EPA and DHA de novo. Results from this study indicate that increasing DOC from low (2 mg DOC/L) to moderate levels (15 mg DOC/L) may increase the importance of the microbial pathway in the trophic transfer of EPA and DHA from basal resources to zooplankton. However, this supplementary transfer of EFA through the microbial food web may not sustain high EPA and DHA levels in zooplankton when lake browning starts to limit primary production (&>15 mg DOC/L).