Objective: The present study aimed to investigate the frequency and antibiotic susceptibility pattern of Gram-negative bacteria (GNB) isolated from surgical site infections (SSIs) in the North of Iran. Results: This cross-sectional study conducted over a two-year period during 2018-2020 on all cases of SSIs who had a positive culture for a GNB. Standard microbiological tests were followed for the bacterial isolation and identification. Antimicrobial susceptibility profiles were determined using disk diffusion method. During the study period, a total of 78 nonduplicated GNB isolated from SSIs. The most prevalent surgical procedures were fracture fixation (37.2%), and tissue debridement (23.1%). Klebsiella isolates showed the highest isolation rate (29.5%) followed by Enterobacter (28.2%), and Acinetobacter (16.7%). Antibiotic susceptibility results showed that Acinetobacter isolates were almost resistant to all of the tested antibiotics, except gentamicin, co-trimoxazole, and meropenem. Enterobacteriaceae isolates showed the lowest resistance against amikacin, co-trimoxazole, and imipenem. Overall, 49 (62.8%) of isolates were multiple drug-resistant (MDR). In summary, a remarkable rate of MDR isolates which showed an increasing trend during recent years is a serious alarm for the management of SSIs caused by GNB. Moreover, the results of regional assessments, provide good epidemiological background for comparing our situation with other regions.