The spread and contamination of antimicrobial-resistant bacteria in ambient waters is an emerging concern in urban, rural, medical, and industrial settings. A large amount of domestic, hospital, and industrial wastewater discharged directly into the rivers through the different channels can turn them into extensive reservoirs of antibiotic-resistant bacteria. In the present study, surface water samples from three collection sites were analyzed, according to different levels of anthropogenic impacts, along the Ozama River, one of the most important rivers in the Dominican metropolitan area, a source of water and food for human consumption. Seventy-six bacterial isolates were selected based on resistance to beta-lactams, using culture media previously enriched with cefotaxime and imipenem. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF) subsequently identified them. The isolates covered 12 genera of bacteria; more than 30% were clinically relevant, and 43% had phenotypes classified as multidrug resistance. A total of 10 (44%) presented resistance. However, only seven presented resistance to 3 or more of the 14 groups of antibiotics, considered to be a multiresistant phenotype, which was sequenced using the high-throughput sequencing technique or New Generation (NGS). This study is part of the initiative to understand the profiles of the dangers of multidrug resistance in the metropolitan and rural areas of the Dominican Republic and its possible implications for human health.