The EU Water Framework Directive foresees the ecological assessment of surface waters against identified pressures. Nutrient loading is the main pressure impairing the ecological quality of lake ecosystems, and aquatic macrophytes are considered good indicators of ecological response. In this study, we statistically assessed different aspects of aquatic plant (macrophyte) diversity in response to different trophic levels in Mediterranean lakes. We used 5690 relevés of aquatic vegetation, distributed over 305 transects, sampled in 18 freshwater lake ecosystems during 2013–2016. Our results show a significant decrease in taxonomic alpha diversity in lakes with a total phosphorus content above 100 μg/L. Syntaxonomic diversity followed the species richness pattern as well. Functional richness decreased along the trophic gradient, while functional dispersion was higher in lakes with high trophic levels. Taxonomic and functional beta partitioning presented changes in assembly processes leading to greater community homogeneity in lakes with higher trophic levels. In summary, we found no redundancy between taxonomic and functional diversity indices. These results provide novel insights into aquatic plant assembly processes of impacted freshwater lakes needed to forward conservation and restoration practices.