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In past studies, the health and ecological risks of PCBs are well established. The impact of low-dose PCBs on aquatic ecosystems for an extended period is a matter of concern in the current era. The application of fatty acids (FAs) as bioindicators of pollution in the freshwater food web is almost unavailable. This study investigated concentrations of 209 PCB congeners, stable isotope levels, and FAs composition in ten freshwater species of Dongtinghu Lake, China. Total PCB congeners (∑PCBs) concentrations were ranged from 4.17 to 38.35 ng/g lipid weight. A total of 84 PCB congeners were detected out of 209 target PCB congeners, particularly PCB101, 118, 138, 153, and 155 found in all samples. The concentrations of 24 PCB congeners increased with trophic levels, but PCB 155 concentrations were consistent throughout trophic levels. The toxic equivalents (TEQ) of dl-PCBs (mostly PCB 126 and 169) also increased with trophic levels Out of total 35 FAs, 21 FAs were significantly positively correlated with 43 PCB congeners. Among FAs, C16:0 was the most abundant and positively correlated with most PCB compounds. Positive correlations between FAs and PCBs indicated that FAs can be used as efficient bioindicators of PCBs pollution in the aquatic food web. [Display omitted] • Stable isotope, fatty acids and PCBs were analyzed in a freshwater lake food web. • A total of 84 PCB congeners were detected out of 209 target PCBs. • Fatty acids were significantly positively correlated with PCBs in the food web. [ABSTRACT FROM AUTHOR]