Macrophytes are good indicators of the river condition because they respond quickly to the changes in physico-chemical parameters, velocity or the level of water. The aim of this study was to investigate the composition and structure of macrophyte vegetation of the middle course of the Mrežnica River and evaluate its biological condition. A total of 14 sites were investigated resulting in 48 recorded macrophyte species. The majority of species were rooted vascular plants that grow in slower and deeper parts of the river and appear in several morphological forms that are classified in Sparganium emersum community. The highest coverage of mosses was found on travertine barriers. They belonged to the Berula-Agrostis community that is significant for travertine waterfalls. This type of habitat had the highest biodiversity and homogeneity of macrophyte vegetation. Nine species were protected at the national level, while one was strictly protected with the status of endangered species (EN), and one was classified in the data-deficient category (DD) according to the criteria of the International Union for Conservation of Nature (IUCN). Based on the analysis of macrophytes, the biological condition of the middle course of the Mrežnica River is classified as “in very good condition” (class 1). This work closely examined the macrophyte vegetation of Mrežnica and can be used for future monitoring, which is important for the development of the management and protection plans.