Enterococcus hirae is a zoonotic Enterococcus species that causes opportunistic infections in both humans and animals and can be transmitted by contact with animals or through contaminated food. The aim of this study was to investigate the importance of E. hirae in broilers with endocarditis, as well as the antimicrobial resistance patterns and genetic relatedness of the isolates. A total of 477 three- to five-week-old broilers were studied during five fattening periods on a farm with mortality due to endocarditis. Endocarditis was observed in 27 chickens (5.66%), and samples were taken for pathological, microbiological, and molecular studies. Lesions were mainly found in the right atrioventricular valve and corresponded with a fibrinous endocarditis. Enterococcus hirae was identified in all cases. Pulsed-field gel electrophoresis results showed clonality among some isolates, with one pulsotype harboring 11 isolates that were found throughout the study. Most of the isolates showed multi-drug-resistant phenotypes. These results confirm that E. hirae is a significant cause of endocarditis in broilers, and suggest that broilers may be important carriers of antimicrobial-resistant E. hirae that might enter into the food chain.