Objective: To investigate the characteristics of pathogen infection and to establish a prediction model of infections in oral squamous cell carcinoma patients undergoing surgery with free flap reconstruction. Methods: The retrospective cohort study consisted of 1 596 patients undergoing tumor resection and free flap reconstruction for oral squamous cell carcinoma from January 2018 to December 2020. According to the postoperative infection, the patients were divided into the infected group (n=154) and non-infected group (n=1 442). The characteristics of pathogens were analyzed in the infected patients. The primary outcome variable was postoperative infection, and Logistic regression was used to determine risk factors of the infection. The prediction model was established and the discriminatory accuracy of the model was evaluated using receiver operating characteristic (ROC) curve. Results: Totally 154 cases were infected in the 1 596 cases undergoing surgery with free flap reconstruction, and the infection rate was 9.65%. The most frequent sites of infection were the surgical wound and respiratory tract. A total of 268 pathogens were isolated and cultured, including 240 strains of Gram-negative bacteria, accounting for 89.55%, mainly Pseudomonas aeruginosa and Klebsiella pneumoniae; 23 strains of Gram-positive bacteria, accounting for 8.58%, mainly Enterococcus faecalis and Staphylococcus aureus; and 5 strains of fungi, accounting for 1.87%. The isolated Pseudomonas aeruginosa had high resistant rate to imipenem and meropenem, and was sensitive to antibiotics, such as ciprofloxacin. The isolated Staphylococcus aureus had high resistant rate to erythromycin and clindamycin, and was sensitive to vancomycin. According to the multivariate Logistic analysis, four independent variables were significantly associated with an increased risk of postoperative infection (P<0.05): clinical N category≥1, the American Society of Anesthesiologists (ASA) grade ≥2, tracheotomy and length of hospital stay >13 d. The prediction model was established based on these factors and the expression of the risk prediction model was as follows: predicted probability value P=1/(1+e^-a), a=-0.803+0.674×(clinical N category ≥1)+0.518×(the ASA grade ≥2)+0.918×(tracheotomy)+1.581×(length of hospital stay >13 d), Hosmer-Lemeshow χ²=10.647, P=0.223, the degree of fitting of the model was good. The area under the ROC curve was 0.818 and 95%CI of the model for predicting infection was 0.789-0.846. Conclusion: Oral squamous cell carcinoma patients undergoing surgery with free flap reconstruction are prone to have a high incidence of postoperative infection and Gram-negative bacteria are the main pathogens causing an infection. The established prediction model is of good predictive effect. Rational antimicrobial use coupled with awareness of infection control measures is paramount to reduce the incidence of postoperative infection in the oral squamous cell carcinoma patients undergoing surgery with free flap reconstruction.