Streptococcus equi subsp. zooepidemicus (S. zooepidemicus), is a β-hemolytic Streptococcus belonging to the Lancefield group C; it is a rare human pathogen, but in horses, it is frequently associated with endometritis. This study aimed to isolate S. zooepidemicus strains, associated with bacterial endometritis in mares, and to define their antimicrobial resistance profile. Twenty-three isolates were recovered from one hundred ninety-six equine uterine swabs (11.7%). Bacterial identification was carried out by Api 20 Strep and confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), while antimicrobial susceptibility testing was performed by disk diffusion method on Muller Hinton agar plates. The antibiotic resistance profiles of the isolates revealed a high percentage of resistance to amikacin (95.6%), ampicillin (73.9%) and tetracycline (69.6%), while ceftiofur and ceftriaxone were highly effective with 82.6% and 78.3% of the isolates inhibited, respectively. An intriguing value of resistance to penicillin (34.8%), which represents the first-choice antibiotic in equine S. zooepidemicus infections, was observed. Furthermore, a high prevalence of multidrug-resistant strains (82.6%) was recorded. Continuous surveillance of this potential zoonotic pathogen and an appropriate antimicrobial stewardship program with the promotion of correct use of antimicrobials, after a proper diagnosis, are needed to allow an effective therapy.