Bacterial contamination of soft tissue in open fractures leads to high infection rates. Pathogens and their resistance against therapeutic agents change with time and vary in different regions. The purpose of this study was to characterize the bacterial spectrum present in open fractures and analyze the bacterial resistance to antibiotic agents based on five trauma centers in East China. A retrospective multicenter cohort study was conducted in six major trauma centers in East China from January 2015 to December 2017. Patients who sustained open fractures of the lower extremities were included. The data collected included the mechanism of injury, the Gustilo-Anderson classification, the isolated pathogens and their resistance against therapeutic agents, as well as the prophylactic antibiotics administered. In total, 1348 patients were included in our study, all of whom received antibiotic prophylaxis (cefotiam or cefuroxime) during the first debridement at the emergency room. Wound cultures were taken in 1187 patients (85.8%); the results showed that the positive rate of open fracture was 54.8% (651/1187), and 59% of the bacterial detections occurred in grade III fractures. Most pathogens (72.7%) were sensitive to prophylactic antibiotics, according to the EAST guideline. Quinolones and cotrimoxazole showed the lowest rates of resistance. The updated EAST guidelines for antibiotic prophylaxis in open fracture (2011) have been proven to be adequate for a large portion of patients, and we would like to suggest additional Gram-negative coverage for patients with grade II open fractures based on the results obtained in this setting in East China.