To what extent co-localized humans and wild animals share commensal Escherichia coli strains and the associated antimicrobial resistance determinants and virulence-associated genes is poorly defined. To study this, over two years (1995 and 1996) we collected fecal Escherichia coli isolates from two communities of chimpanzees that inhabit the Kibale National Park in Uganda and compared them to contemporaneous fecal E. coli isolates collected from villagers living at the boundary of the preserve and park workers (139 total isolates). We found that antimicrobial resistance was most prevalent among park workers (25 subjects), intermediately prevalent among Kanyawara community chimpanzees (which have human contact: 33 subjects), and least prevalent among villagers (6 subjects) and Ngogo community chimpanzees (which lack direct human contact: 20 subjects). Molecular analysis of the 139 isolates to assess the frequency and distribution of 54 molecular characteristics (phylogenetic group markers and virulence-associated genes) showed that strains from both groups of chimpanzees (Kanyawara, n = 64; Ngogo, n = 28) were more similar to strains from villagers (n = 16) than to strains from park workers (n = 31). Genes associated with extraintestinal disease in humans such as fimbrial and afimbrial adhesins, hemolysin, toxins, and iron uptake systems were, paradoxically, most prevalent among chimpanzee isolates. By multi-dimensional scaling, the closest between-species population similarity for molecular characteristics was between strains from Kanyawara chimpanzees and those from villagers. These data support the potential sharing of transmissible resistance markers between chimpanzees and humans in close contact, whereas similarities in commensal strains’ genetic background is more related to shared habitat and host species.