To assess if environmental differences other than water quality may affect the outcome of the Benthic Quality Index, a comparison of the application of four different methods (Benthic Quality Index—BQIES, Lake Habitat Modification Score—LHMS, Lake Habitat Quality Assessment—LHQA and Organisation for Economic Co-operation and Development—OECD) used to classify the lake ecological and hydro-morphological status of 10 Italian lakes was performed. Five lakes were natural and five were reservoirs belonging to both Alpine and Mediterranean Ecoregions. The 10 lakes were sampled using the Water Framework Directive compliant standardized national protocol, which includes sampling soft sediment in the littoral, sublittoral and deep layers along transects with a grab of 225 cm2 during spring and autumn. The application of Generalised Linear Mixed Effect Models both at the lake level and at the single station of each lake highlighted that, at the lake level, no significant correlations existed between any couple of hydro-morphological, ecological and trophic status assessments, with each metric representing a different facet of human impact on the environment. At the single site level, we found significant effects of depth on the metrics of biodiversity. The best approximation of single-site macroinvertebrates diversity among the metrics of overall lake quality was with the LHMS, but not with the BQIES. Our hypotheses that lake macroinvertebrates assemblages depend also on other potential confounding variables of habitat degradation and intrinsic differences between lakes were confirmed, with depth playing a major role. Therefore, the assessment of lakes with different depths may produce different whole-lake BQIES values, only because of the effect of depth gradient and not because of differences in lake quality.