This study aimed to document antimicrobial resistance patterns of bacteria frequently isolated at a referral equine hospital between 2020 and 2022 and to compare these results to those of studies carried out in previous decades at the same reference center. Using the Kirby-Bauer method, 340 tests were completed on bacterial isolates and compared to 233, 255, and 396 tests carried out in 1986 to 1988, 1996 to 1998, and 2007 to 2013, respectively. Data were analyzed with X 2 tests for trends, followed by pairwise Fisher tests and Bonferroni corrections. Increasing resistance to at least 1 antibiotic was observed for 5 of the 6 bacteria studied, including increasing resistance to enrofloxacin for Escherichia coli, Pseudomonas spp., Enterobacter spp., and staphylococci, and to ceftiofur for Enterobacter spp. and staphylococci. Resistance of Actinobacillus spp. to ampicillin and penicillin decreased over time, whereas resistance of Streptococcus equi subsp. zooepidemicus to penicillin was not observed. Bacteria isolated from horses had increasing resistance to antibiotic classes considered critically important for human health. However, penicillin remained an appropriate 1st-line antibiotic for Actinobacillus spp. and Streptococcus equi subsp. zooepidemicus in our area.