Wildlife may be an important reservoir of antibiotic-resistant bacteria and resistance genes. In this pilot study, the prevalence and patterns of antimicrobial resistance in Escherichia coli cultured from wild herring gull (Larus argentatus) feces and human wastewater at Cape Cod, Massachusetts, USA, was compared. Antimicrobial susceptibility was tested using Kirby–Bauer disk diffusion with seven antimicrobial agents. A high proportion of antimicrobial agent-resistant E. coli isolates (59.2%) were detected in wastewater samples compared with a lower prevalence of 17.5% in gull feces. In addition, there was a large proportion of isolates with intermediate susceptibility (93.0%) in gull feces. Although similar resistance patterns and shared resistance genes suggest possible wastewater contamination of the local environment, the relatively low frequency of resistance and high prevalence of intermediate susceptibility detected in E. coli cultured from gull feces depict a complex model of antimicrobial resistance among E. coli strains of wildlife origin.