Imaging methods including conventional radiography, computed tomography, fistulography and ultrasonography have been used to detect signs of bone damage caused by osteomyelitis. However, microbiological cultures are still the routine method, more accurate for the definitive diagnosis of osteomyelitis, and also help determine the treatment choice. Therefore, the aim of this study was to evaluate retrospectively, for a period of 12 years, microbiological cultures of dogs with clinical suspicion of osteomyelitis, as well as determine the antibiotic susceptibility profile. The isolated microorganisms were identified based on morphology and staining, biochemical tests and culture using 32 antimicrobials. In vitro microbial sensitivity of isolates were performed by disk diffusion method technique. Fifty-one microbiological cultures were evaluated, 87% positives and 13% negatives. Nineteen different microorganisms were isolated, 18 bacteria and one fungi. The most frequently isolated microorganisms were Staphylococci and E. coli followed by streptococci, enteric bacteria, actinomycetes, and anaerobic bacteria. The most effective drugs against isolated bacteria were amoxicillin and clavulanate potassium (79.17%), ceftriaxone (67%), and chloramphenicol (65%). The isolated bacteria showed high resistance rate against azithromycin (80%), penicillin (58.6%), and clindamycin (58.6%). In conclusion, the most prevalent bacteria were beta-hemolytic Staphylococcus and Escherichia coli, and amoxicillin-clavulanate had a high sensitivity rate and low resistance compared to the other tested antibiotics.