Antimicrobial resistance (AMR) is a critical challenge of the 21st century for public and animal health. The role of host biodiversity and the environment in the evolution and transmission of resistant bacteria between populations and species, and specifically at the wildlife-livestock-human interface, needs to be further investigated. We evaluated the AMR of commensal Escherichia coli in three mammalian herbivore species—impala (Aepyceros melampus), greater kudu (Tragelaphus strepsiceros), and plains zebra (Equus quagga)—targeting populations living under two conditions: captivity (French zoos) and free ranging (natural and private parks in Zimbabwe). From 137 fecal samples from these three host species, 328 E. coli isolates were isolated. We measured the AMR of each isolate against eight antibiotics, and we assessed the presence of AMR genes and mobile genetic element class 1 integrons (int1). Isolates obtained from captive hosts had a higher probability of being resistant than those obtained from free-ranging hosts (odds ratio, 293.8; confidence interval, 10– 94,000). This statistically higher proportion of AMR bacteria in zoos than in natural parks was especially observed for bacteria resistant to amoxicillin. The percentage of int1 detection was higher when isolates were obtained from captive hosts, particularly captive impalas. Ninety percent of bacterial isolates with genes involved in antibiotic resistance also had the int1 gene. The sul1, sul2, blaTEM, and stra genes were found in 14, 19, 0, and 31%, respectively, of E. coli with respective antibiotic resistance. Finally, plains zebra carried AMR significantly more often than the other species.