Benthic diatoms inhabiting littoral zones are regarded as promising bioindicators in lakes. Some pre-standard protocols were proposed and suggested to collect a single site per lake to assess its overall quality. In large lakes, such as Lake Geneva, a spatial heterogeneity of diatom assemblages was suspected, because of differing human pressures. Thirty-five sites were sampled along the shoreline in locations showing various population densities and crossed by rivers flowing into the lake. Different assemblages were found; one corresponded to more oligotrophic areas and was mostly situated on the shoreline next to low population densities; it was mostly composed of low-profile diatoms. A second one corresponded to more eutrophic areas and was mostly situated near densely inhabited areas; it was composed of many high-profile diatoms. A third one was in close vicinity to river inlets, with high abundances of motile diatoms. This last assemblage was spatially restricted to the river plume. After testing several diatom indices, one showed better capacities to assess nutrient level (EPI-L). If we avoid areas near river inlets, the ecological quality assessed with diatom indices ranged from moderate to high ecological status depending on the vicinity situated more or less near densely populated areas.