Abstract Objective To evaluate bacterial isolates from corneal stromal ulcerations in dogs and cats in the Netherlands, review their antibiotic susceptibility, determine whether recent topical treatment affected bacterial culture results, and investigate whether (multi‐drug) resistance patterns changed over time. Animals studied Client‐owned dogs and cats were diagnosed with corneal stromal ulceration at the Utrecht University Clinic for Companion Animals between 2012 and 2019. Procedures Retrospective analysis. Results In total, 163 samples were collected from 122 dogs (130 samples) and 33 cats. Positive cultures were obtained from 76 canine and 13 feline samples (59% and 39%, respectively) and included Staphylococcus (42 in dogs, 8 in cats), Streptococcus (22 in dogs, 2 in cats), and Pseudomonas (9 in dogs, 1 in cats) species. Significantly fewer positive cultures were found in dogs and cats previously treated with topical antibiotics ( χ 2 = 6.52, p = .011 and χ 2 = 4.27, p = .039, respectively). Bacterial resistance to chloramphenicol was more common in dogs previously treated with chloramphenicol ( χ 2 = 5.24, p = .022). The incidence of acquired antibiotic resistance did not increase significantly over time. In dogs, the incidence of multi‐drug‐resistant isolates increased significantly between 2012–2015 and 2016–2019 (9.4% vs. 38.6%, p = .0032). Conclusions Staphylococcus, Streptococcus , and Pseudomonas species were the most common bacteria associated with canine and feline corneal stromal ulcerations. Previous treatment with antibiotics affected bacterial culture results and antibiotic sensitivity. Although the overall incidence of acquired antibiotic resistance did not change over time, the incidence of multi‐drug‐resistant isolates in dogs increased over an 8‐year period.