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Antimicrobial resistance (AMR) is a critical global issue affecting public and animal health. The overuse of antibiotics in human health, animal production, agriculture, and aquaculture has led to the selection of antibiotic-resistant strains, particularly in Gram-negative bacteria. Mutations and horizontal gene transfer play a significant role in the development of antimicrobial resistance, leading to the reduced efficacy of current antibiotics. Today, AMR in bacteria and antibiotic-resistance genes (ARGs) are increasingly recognized in multiple environmental sources, including recreational and irrigation waters. This study aims to identify Gram-negative bacteria from surface aquatic reservoirs in southern Chile and assess their susceptibility to clinically relevant antibiotics. Water samples were collected from four lakes, five rivers, one waterfall, and one watershed in southern Chile to isolate environmental Gram-negative bacilli (GNB). API-20E and MALDI–TOF were employed for bacterial identification. Kirby–Bauer disc diffusion tests and multiplex PCR were performed to determine their susceptibility profile. A total of 26 GNB strains were isolated from environmental water samples, predominantly belonging to the Pseudomonas (n = 9) and Acinetobacter (n = 7) genera. Among these strains, 96.2% were resistant to ampicillin and cefazoline, while 26.9% and 34.6% showed resistance to ceftazidime and cefepime, respectively. Additionally, 38.5% exhibited resistance to colistin. Two Enterobacter cloacae strains obtained from Cachapoal River (sixth region) and Villarrica Lake (ninth region), respectively, presented a multidrug-resistant (MDR) phenotype and carried at least two extended-spectrum β-lactamase (ESBL) genes. Thus, antibiotic-resistant GNB and ARGs were found in natural water reservoirs, raising concerns about the dissemination of resistance determinants among potentially pathogenic bacteria in environmental microbial communities. [ABSTRACT FROM AUTHOR]