The aim of the study was to identify the bacterial profile and antimicrobial susceptibility from surgical site cultures in queens and bitches undergoing ovariohysterectomy via ventral midline and lateral flank approaches. Healthy 22 queens and 21 bitches were assigned randomly either ventral midline or lateral flank approach for routine ovariohysterectomy. Surgical site samples were collected before the surgery and on the $1^{st}, 2^{nd} ,and 3^{rd}$ days after the surgery for microbiological analysis. A total of 70 different strains were isolated from 50 (29%) of 172 samples. In both queens and bitches the total number of bacteria isolated from the midline approach (n = 50) was found to be higher than in the flank approach (n = 20). The most frequently isolated bacteria were Staphylococcus intermedius group (SIG) (38.5%), followed by Enterococcus faecium (10%) and Staphylococcus lentus (8.5%). Of the isolated Staphylococcus spp. strains were positive 64% for beta-lactam resistance while 52% of that strains were also methicillin-resistant. Multidrug resistance to methicillin, beta-lactamase, and clindamycin was determined in two Staphylococcus spp. isolates. A high level of streptomycin resistance was observed in three Enterococcus faecium isolates. Overall, this study revealed that choosing the surgical site for ovariohysterectomy affected the bacterial profile and more than half of the isolates were resistant to antimicrobials. Thus, the lateral flank approach might be better than the ventral midline approach to prevent possible complications such as a surgical site infection in queens and bitches undergoing ovariohysterectomy.