The increasing antimicrobial resistance (AMR) of pathogens is a significant threat to human and animal health, but it is also an environmental challenge for water resources. The present study aimed to quantify heterotrophic bacteria resistant to five groups of antibiotics (ABs) in a selected Yantra River stretch (including its tributary, the Belitsa River); to assess AMR prevalence among Enterobacteriaceae; and to assess the impact of urban effluents or rural runoff on AMR prevalence along the river course at eight sampling points. Culture-dependent methods were used in a population-based study of total AMR and for AB susceptibility testing of Enterobacteriaceae isolates. The data reveal significant differences in AMR dissemination and a lower (up to 10%) proportion of different types of antibiotic-resistant bacteria (ARB) in the Yantra River water compared to the Belitsa River (up to 20%). The incidence of resistant Enterobacteriaceae isolates was in the range of 1% to gentamicin to 36% to ampicillin, including multidrug resistance of 19%, and different AMR patterns of isolates from each river. The prevalence of AMR among aquatic bacteria highlights the need for adequate waste water treatment and for management, monitoring and control of treatment processes to limit anthropogenic pressure through discharge of untreated or incompletely treated waste water and to ensure the ecological well-being of receiving waters.