This study aimed to investigate the prevalence and antibiotic resistance of Staphylococcus aureus isolates from Pangasius fish (Pangasianodon hypophthalmus) and the hands of fish processors in fish processing plants. The results showed that 16 of 90 Pangasius fish (18%) and 6 of 54 fish processing handlers’ samples (11%) harbored coagulase-positive for S. aureus. A high antibiotic resistance (68%) was observed for ampicillin, while 100% susceptibility for oxacillin, cefazolin, cefmetazole, flomoxef, imipenem, arbekacin, fosfomycin, and sulfamethoxazole-trimethoprim were recorded for both fish and hand samples. Remarkably, only one isolates from the fish was classified as methicillin-resistant S. aureus, whereas no isolates from the handlers were found. Among the total S. aureus isolates, the multidrug resistance rate was nearly 41%, posing a hazard to public health concerns. The obtained results may provide valuable information about the actual state of S. aureus contamination for efficient control in fish processing plants.