According to the requirements of the European Water Framework Directive, the Member States of the European Union are obliged to assess and report on the ecological potential of heavily modified and artificial water bodies; water reservoirs on rivers were also designated among them. The objective of this study was to gain more knowledge about macrophyte assemblages in reservoirs in Slovakia, where it was necessary to start analyses leading to the ecological assessment. The research was carried out in 14 multipurpose reservoirs during the vegetation seasons 2008–2010. Analyses focused on the determination of species composition considering the similarity between reservoirs, the impact of selected environmental variables on species composition and evaluation of the Macrophyte Biological Index for Lakes (IBML) in relation to its use for ecological assessment. In total, 60 taxa of macrophytes were identified. More than 90% of all determined taxa are indicators of IBML. The statistical analysis performed was based on the study of macrophyte assemblages and environmental variables and gave the following results: i) based on species composition, two main clusters of reservoirs were identified respecting altitude (reservoirs at an altitude above and less than 300 m a.s.l.) and affiliation to phytogeographical ecoregion (reservoirs in Pannonian lowland and Carpathians); ii) water temperature, followed by dissolved oxygen and chemical oxygen demand, were found to be the main environmental variables influencing the composition of macrophyte assemblages using DCA analysis. Water temperature and phosphates were determined to be the variables responsible for species composition using CCA analysis; iii) differences of the mean IBML values between clusters corresponded with the results of cluster analysis. A significant correlation was found between IBML and two variables: conductivity and alkalinity. Based on the results, it is recommended to use the IBML for analyses leading to the assessment of ecological potential based on biological quality elements.