Dystrophic lakes are widespread in temperate regions and intimately interact with surrounding terrestrial ecosystems in energy and nutrient dynamics, yet the relative importance of autochthonous and allochthonous resources to consumer production in dystrophic lakes remains controversial. We argue that allochthonous organic matter quantitatively dominates over photosynthetic autotrophs in dystrophic lakes, but that autotrophs are higher in diet quality and more important for consumers as they contain essential polyunsaturated fatty acids (PUFA). In a field study, we tested the hypotheses that (1) autochthonous primary production is the main driver for consumer production, despite being limited by light availability and low nutrient supplies, and greater supply of allochthonous carbon, (2) the relative contribution of autotrophs to consumers is directly related to their tissue PUFA concentrations, and (3) methane-oxidizing bacteria (MOB) provide an energy alternative for consumers. Pelagic and benthic consumer taxa representing different trophic levels were sampled from five dystrophic lakes: isopod Asellus aquaticus, megalopteran Sialis lutaria, dipteran Chaoborus flavicans, and perch Perca fluviatilis. Based on carbon and nitrogen stable isotopes, the relative contributions of autochthonous (biofilms and seston) and allochthonous (coarse particulate and dissolved organic matter) resources and MOB to these taxa were 47-79%, 9-44% and 7-12% respectively. Results from fatty acid (FA) analyses show that the relative omega3-FA and PUFA concentrations increased with trophic level (Asellus < Sialis and Chaoborus < Perca). Also, eicosapentaenoic-acid (EPA), omega3-FA and PUFA concentrations increased with the autochthonous contribution in consumers, i.e., a 47-79% biofilm and/or seston diet resulted in tissue EPA of 4.2-18.4, omega3 FAs of 11.6-37.0 and PUFA of 21.6-61.0 mg/g dry mass. The results indicate that consumers in dystrophic lakes predominantly rely on energy from autotrophs and that their PUFA concentrations are dependent on the relative contribution of these autochthonous resources. The limited energy support from MOB suggests they are not negligible and are potentially an integral part of the food webs. Our findings show that autochthonous resources are the main driver of secondary production even in dystrophic lakes and offer new insights into the functioning of these ecosystems.