The aim of the study is to identify levels of per- and polyfluoroalkyl substances and brominated flame retardants in Prague waters, to potentially identify the main sources of pollution by the monitored substances in the Prague waters and generally contribute to a greater knowledge of pollution by the monitored substances in the Czech Republic. A total of seven river water samples, three sediment samples, and four fish samples from Prague streams, a pond, and the Vltava River were analysed for content of per- and polyfluoroalkyl substances and brominated flame retardants in the laboratory of the Department of Food Analysis and Nutrition of the University of Chemistry and Technology in Prague. According to the data collected, pollution of surface waters in Prague by per- and polyfluoroalkyl substances and brominated flame retardants is ubiquitous.