The lake littoral sediment is exposed to a large array of contaminants that can exhibit significant spatial variability and challenge our ability to assess contamination at lake scale. In this study, littoral sediment contamination was characterized among ten different sites in a large peri-alpine lake (Lake Bourget) regarding three groups of contaminants: 6 heavy metals, 15 polycyclic aromatic hydrocarbons and 7 polychlorinated biphenyls. The contamination profiles significantly varied among sites and differed from those previously reported for the deepest zone of the lake. An integrative approach including chemical and biological analyses was conducted to relate site contamination to ecological risk. The chemical approach consisted in mean PEC quotient calculation (average of the ratios of the contaminants concentration to their corresponding Probable Effect Concentration values) and revealed a low and heterogeneous toxicity of the contaminant mixture along the littoral. Biological analysis including both laboratory (microcosm assays) and in situ (Acetylcholine Esterase (AChE) and Glutathione S-Transferase (GST) activity measurements) experiments highlighted significant differences among sites both in the field and in laboratory assays suggesting a spatial variation of the biota response to contamination. Linear regressions were performed between mean PEC quotients and biological results to assess whether littoral ecological risk was explained by the contamination profiles. The results highly depended on the study benthic or pelagic compartment. Regarding autochthonous Corbicula fluminea, no significant relationship between mean PEC quotients and biomarker activity was found while a significant increase in AChE was observed on autochthonous chironomids, suggesting different stress among benthic organisms. Both AChE and GST in caged pelagic Daphnia magna showed a significant positive relationship with mean PEC quotients. This study underlines the importance of accounting for spatial variations in lake littoral sediment contamination and the need for performing an integrative approach coupling chemical, field and laboratory analyses to assess the ecological risk.