Antimicrobial resistance (AMR) is an increasingconcern to global health, complicating the treatment of infections and contributing to millions of deaths worldwide. Aquatic ecosystem act as reservoirs for antibiotic resistant bacteria and antibiotic-resistant genes due to discharge of industrial and domestic effluents directly into the waterbodies.While A. baumannii, amember of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) group is a well-known pathogen for hospital-acquired infections (HAIs) but simultaneouslyother species of Acinetobactersuch as A. juniiisalso emerging as opportunistic pathogens especially in multi-drug resistant (MDR) infections. In this study, we isolated and identified bacterial isolates from the Kosi River in Uttarakhand, a relatively unpolluted freshwater sourcecompared to urban water bodies. Out of 25 isolates, 24 were identified as Acinetobacter junii through biochemical characterization and 16S rRNA gene sequencing. These isolates were screened for multidrug resistance using the disc diffusion method against 13 different antibiotics belonging to six different classes, and broth microdilution assay was performed for selected isolates for determining their susceptibility to PolymyxinB and Colistin. Notably, 100% of the A. junii isolates were resistant to Cefepime, 33% to Cefoxitin, and 4% to both Ceftazidime and Amikacin. The detection of MDR A. junii in a pristine environment underscores the growing environmental dimension of AMR. This study suggests that natural water bodies may serve as silent reservoirs of resistance genes. The findings reinforce the urgent need for integrated environmental surveillance, stricter control on antibiotic contamination, and global commitment to antimicrobial stewardship to safeguard both environmental and public health.