This study investigated the impact of human activity on antimicrobial resistance (AMR) in the Galapagos marine ecosystem. We focused on San Cristobal Island, which has experienced rapid urbanization and tourism pressure, and found significant wastewater contamination in the marine environment. Long-read metagenomic sequencing revealed a stark shift in genera and a higher count of antibiotic resistance genes at wastewater-associated sites. Long-read sequencing and de novo assembly of bacterial genomes and plasmids from multidrug-resistant Escherichia coli revealed frequent and rapid reassortment of antimicrobial resistance genes on plasmids to generate unique resistance profiles. These data highlight potential threats to human and animal health.