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<i>Escherichia coli</i> is the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24 <i>E. coli</i> strains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%) <i>E. coli</i> strains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, with <i>fimH</i> detected in all strains, and followed by <i>kpsMTII</i> (11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on plates <i>versus</i> strains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight into <i>E. coli</i> strains associated with mares’ infertility. These results broaden the knowledge of <i>E. coli</i> and, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in mares.