Antimicrobial resistance in natural environments poses an increasing threat to public health and biodiversity conservation. This study investigated antimicrobial resistance and biofilm formation in bacteria isolated from Inia araguaiaensis, an Amazonian endemic dolphin species. Skin lesion samples were collected from eight individuals in the Mocajuba region, Pará, Brazil. Bacterial isolation was performed on Brain Heart Infusion agar and Mannitol Salt agar. Species identification was carried out using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antimicrobial susceptibility testing was conducted by disk diffusion following. Resistance genes were detected by polymerase chain reaction, and biofilm formation was assessed using the crystal violet staining method. A total of 19 bacterial isolates were recovered, including Staphylococcus spp. (n = 14) and Mammaliicoccus sciuri (n = 5). The most frequently identified species were Staphylococcus aureus (4/19; 21.1%), S. warneri (4/19; 21.1%), and S. epidermidis (4/19; 21.1%). A high prevalence of penicillin resistance was observed (18/19; 94.7%), followed by resistance to erythromycin, tetracycline, and sulfamethoxazole-trimethoprim (each 4/19; 21.1%), and gentamicin (3/19; 15.8%). Four isolates (4/19; 21.1%) exhibited a multidrug-resistant (MDR) profile. Genotypic screening revealed the presence of the blaZ gene in 8/19 isolates (44.4%), followed by mecA, msrA, and norC (each 2/19; 10.5%). The tetracycline resistance genes tetL and tetM were detected in 3/19 (15.8%) and 1/19 (5.3%) isolates, respectively. Biofilm formation was detected on 15/19 (78.9%) of the isolates, of which 4/15 (26.7%) were classified as strong producers, 1/15 (6.7%) as moderate, and 10/15 (66.7%) as weak producers. These findings support the recognition of river dolphins as potential sentinels for antimicrobial resistance in aquatic ecosystems and reinforce the importance of integrating microbiological monitoring into broader conservation efforts under the One Health perspective.