Background <p>Conjunctivitis is a common clinical disorder in cats and dogs, frequently associated with bacterial and fungal infections. Accurate identification of pathogens and evaluation of their antimicrobial resistance profiles are essential for effective treatment.</p> Methods <p>Bacterial and fungal agents were analyzed using cultural and MALDI-TOF MS methods in conjunctival swab samples taken from 59 domestic animals (53 cats and 6 dogs) diagnosed with conjunctivitis and presented to veterinary clinics in Çankırı, Türkiye, during the period from 2024 to 2025.</p> Results <p>Among the 59 conjunctival swab samples collected from cats and dogs with conjunctivitis, 20 (33.9%) were positive for bacterial culture. Of these, 17 positive cultures were detected in 53 cat samples (32.08%), whereas 3 were detected in 6 dog samples (50.0%). No growth was observed in cultures for anaerobic and Mycoplasma agents, and no evidence of fungal agents was found. Multiple bacterial isolates were isolated in six (10.17%) animals [5 cats (9.43%), 1 dog (16.67%)]. Species identification by MALDI-TOF MS revealed a total of 16 different bacterial species with high confidence scores (≥2.0) across both animal species. The most frequently isolated strains were Staphylococcus epidermidis (n= 7), Corynebacterium mucifaciens (n= 5), Psychrobacter spp. (n= 5), and Streptococcus lutetiensis (n= 4). Rare strains such as Brachybacterium muris (n= 1), Micrococcus luteus (n= 1), and Pseudomonas aeruginosa (n= 1) were also detected. In cats, S. epidermidis, Corynebacterium spp., Psychrobacter spp., and Streptococcus hyovaginalis were the most frequently isolated strains. In dogs, Streptococcus canis, Enterococcus durans, Corynebacterium mucifaciens, Micrococcus luteus, and Staphylococcus hominis ssp. hominis were detected. Antibiotic susceptibility of the isolates was evaluated according to EUCAST and CLSI criteria and interpreted based on the inhibition zone diameter. Of the 37 bacterial isolates recovered, 32 (86.49%) exhibited multidrug-resistant (MDR) profiles. Particularly noteworthy were the extensive MDR patterns observed in Enterococcus durans, Enterococcus faecium, Psychrobacter sanguinis, Streptococcus hyovaginalis, Escherichia coli, and Pseudomonas aeruginosa isolates, which showed resistance to 10-12 antimicrobial agents from multiple classes, including penicillins, tetracyclines, and cephalosporins.</p> Conclusion <p>Although no fungal agents were recovered, the study demonstrates that the bacterial species isolated from cats and dogs with conjunctivitis are highly diverse and that multidrug-resistant (MDR) isolates are common. The antimicrobial susceptibility results provide valuable information on the resistance profiles of microorganisms associated with conjunctivitis cases in the study population. In addition, the detection of certain microorganisms with zoonotic potential underscores the importance of ongoing surveillance from a veterinary public health perspective.</p>