The trophic state of a freshwater ecosystem reflects its environmental quality. This is why several trophic indicators have been developed for such water bodies based on chemical, physical and biological parameters. Apart from that, there are several biotic elements which can be used in accessing the environmental condition of a freshwater ecosystem. Zooplankton organisms are important elements of the structure and function of lakes and are considered useful indicators of alterations in their trophic dynamics and ecological state related to changes in nutrient loading and climate. In accordance to the above, the present study is an attempt to assess the trophic condition of the largest lake in Greece (Lake Trichonis) through the investigation of the physicochemical elements, along with the biotic indications provided by a three-year study of the lake’s zooplankton. The present results, compared with previous studies conducted between 15 and 25 years before, showed that there was an increase in the maximum values of the concentrations of chlorophyll-α and nutrients, while there was a decrease in water transparency. The implementation of Carslon’s trophic state index (TSI) revealed that Lake Trichonis still remains an oligo-to mesotrophic ecosystem as it was in the past. However, although the zooplankton investigation showed several features that are common in oligotrophic lakes, there are certain eutrophic characteristics of the zooplankton community (e.g. abundance variation pattern, indicator species, seasonal succession of cladocerans) pointing out a different state of the ecosystem in comparison to the past. In conclusion, the use of a biotic element like zooplankton revealed that Lake Trichonis is experiencing a transitional condition towards the eutrophic state and points out the necessity for constant inspection and monitoring of this ecosystem.