Antimicrobial resistance (AMR) poses a global challenge, particularly with regard to the ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which are associated with severe and often fatal infections. This study aimed to assess the occurrence of ESKAPE pathogens and Serratia marcescens in hospital wastewater effluents and influent samples from the wastewater treatment plant (WWTP) in Panama City. The methodology involved enriching cell cultures, utilizing selective media for growth, and conducting biochemical assessments using the Vitek® 2 automated system for detection and susceptibility profiling. Molecular confirmation of resistance genes was achieved through end-point PCR analysis. A total of 75 strains each of A. baumannii and P. aeruginosa, 50 strains of S. aureus, 45 strains of Enterococcus sp., 10 strains of S. marcescens, and 75 strains of Klebsiella spp. were identified. In terms of genotypic profiles, it was found that 3% of the P. aeruginosa strains possessed the blaVIM gene, while a significant majority, 97%, carried blaOXA-48. Among Klebsiella spp., 13% contained blaKPC, 3% had blaNDM, 13% showed presence of blaCTX-M, 6% had blaVIM, and 8% were positive for the pilV gene. Additionally, the mecA gene was identified in 20% of S. aureus strains classified as MRSA. These findings underscore the significance of wastewater as a crucial reservoir for resistance genes and highlight a major public health concern in Panama.