A new multi-criteria method of evaluation and assessment of the ecological status of lakes is proposed. It is based on macrophytes analysis integrated with geomorphological, landscape and catchment sources of threats. A total of 22 lakes in the Transboundary Biosphere Reserve ‘West Polesie’ (Poland) were investigated along trophic (available nutrients) and human pressure gradients, testing the proposed method with ESMI and TRS indices. Therefore, the present indexation included 22 criteria (i.e., catchment land use, phytolittoral area, number of plant species) concerning three different assessing zones (lakeshore, littoral and surrounding area), and provided a five-class ecological classification. The proposed index, in addition to the general ecological conditions assessment of lakes, allows to point out a zonal evaluation, identifying the most critic zones in terms of ecological status. The proposed method can be universally adapted for any type of lakes, regardless of their geographical characteristics. It can be applied to system monitoring, and to support lakes biodiversity, functionality, conservation, restoration, water protection and uses, as well as water, territory and landscape management actions.