Accredited zoos and animal parks play an important role in animal health research and conservation, providing important insights on matters of public health including zoonotic infectious diseases and antimicrobial resistance (AMR). The emergence and spread of AMR is a complex phenomenon that jeopardizes human and animal health and also threatens the long-term survival of endangered species. The presence of β-lactamases in clinical isolates is particularly significant as they can jeopardize the efficacy of critically important antimicrobials. Although the presence of β-lactamases and extended-spectrum β-lactamases (ESBLs) producing Enterobacteriaceae in zoo animals has been reported, data are not available for northern European countries. In addition, few data are available on phylogenetic grouping of Escherichia coli isolated from zoo animals that can provide additional information on the host–bacterium relationship and on the pathogenicity of isolates. This study aimed to characterize fecal E. coli isolated from 33 healthy zoo animals from 22 species in Ireland, using conventional and molecular microbiological methods. All E. coli isolates were ampicillin resistant, but combined resistance to amoxicillin and clavulanic acid was not detected. Three E. coli isolates sampled from one Amur tiger, one Bornean orangutan, and one Southern white rhino were multidrug resistant, and blaTEM was detected in E. coli recovered from the Amur tiger and the Bornean orangutan. Other β-lactamases, including ESBLs and AmpCs and plasmid-mediated mcr-1 and mcr-2, were not detected. Overall, E. coli isolates investigated were susceptible to the majority of the antimicrobials tested, and only two animals shed E. coli carrying β-lactamase–encoding genes. The majority of isolates belonged to phylogenetic group B1. The screening of the AMR phenotype and genotype of zoo animal E. coli provides useful data that is relevant to antimicrobial stewardship in the zoo veterinary services and relevant to the bank of knowledge needed for tackling AMR.