Aquatic ecosystems subjected to anthropic pressures are likely hotspots for emergence or dissemination of antimicrobial resistant bacteria. The city of Montpellier is located on a Mediterranean climate watershed that undergoes strong demographic pressures. The aim of the study is to explore antimicrobial resistance, particularly those of clinical concern, in urban rivers flowing in this urban area. The method developed herein to explore antimicrobial resistance is based on cultural and molecular approaches completed by hydrological, hydrogeological, climatic, and physico-chemical data. Hospital vicinity and urbanization density significantly increase cultivable bacterial community, fecal bacteria from human origin, and prevalence of β-lactamases and extended-spectrum β-lactamases encoding-genes without an increase in 16S rDNA gene abundance. A total of 22 multidrug Enterobacterales have been isolated. All Escherichia coli (n = 10) and Klebsiella pneumoniae (n = 6) isolated on a made-house media carried β-lactamases genes, bla_CTX-M being the most prevalent (87%), followed by bla_TEM (56%) and bla_SHV (37%), 56% of these strains carrying two or three of these genes. In urban settings, water quality and infectious risk are generally linked to wastewater treatment plants effluents. This study shows that running waters in urbanized area contribute to the dissemination of antimicrobial resistance, making these environments a reservoir for resistant bacteria with important consideration.