Aquatic vegetation is an essential component of lake ecosystems, used as a biological indicator for in situ monitoring within the Water Framework Directive. We developed a hierarchical object-based image classification model with multi-seasonal Sentinel-2 imagery and suitable spectral indices in order to map the aquatic vegetation in a Mediterranean oligotrophic/mesotrophic deep lake; we then applied the model to another lake with similar abiotic and biotic characteristics. Field data from a survey of aquatic macrophytes, undertaken on the same dates as EO data, were used within the accuracy assessment. The aquatic vegetation was discerned into three classes: emergent, floating, and submerged aquatic vegetation. Geographic object-based image analysis (GEOBIA) proved to be effective in discriminating the three classes in both study areas. Results showed high effectiveness of the classification model in terms of overall accuracy, particularly for the emergent and floating classes. In the case of submerged aquatic vegetation, challenges in their classification prompted us to establish specific criteria for their accurate detection. Overall results showed that GEOBIA based on spectral indices was suitable for mapping aquatic vegetation in oligotrophic/mesotrophic deep lakes. EO data can contribute to large-scale coverage and high-frequency monitoring requirements, being a complementary tool to in situ monitoring.