Background: Infections caused by Staphylococcus aureus strains are a major public health challenge worldwide, especially in specialized burn hospitals. Infections caused by S. aureus account for more than 50% of burn-related deaths. Objectives: Since data on characteristics of these isolates are not sufficient, the current study aimed to assess the prevalence of resistance to antibacterial agents and to analyze the distribution of biofilm, and adhesion encoding genes among S. aureus strains isolated from burn patients in Motahari Hospital, Tehran, Iran. Methods: A total of 83 S. aureus strains were collected from burn wounds of patients admitted to a referral burn center in Tehran for 10 months. In vitro antibacterial susceptibility of isolates was evaluated using the Kirby-Bauer disk diffusion method. Strains were subjected to polymerase chain reaction (PCR) to determine the presence of nucA, mecA, ebps, cna, bbp, fnbA, fnbB, clfA, and clfB genes. Results: The highest frequency of resistance was found to cephalexin and cefoxitin (87.9%), followed by clindamycin (75.9%), erythromycin (72.3%), and ciprofloxacin (60.2%). Five resistance patterns were identified in which cephalexin, cefoxitin, clindamycin, erythromycin, and ciprofloxacin had the most predominant resistance profile (36.1%). Biofilm gene detection indicated a markedly high prevalence of cna (74.7%), clfB (54.2%), clfA (50.6%), fnbA (42.1%), ebp (13.2%), and fnbB (12%). Six different biofilm genetic patterns were identified, wherein clfA, clfB, fnbA, ebp, and cna (30.1%), clfA, clfB, fnbA, fnbB, ebp, and cna (12%), and clfA, clfB, and cna (8.4%) were the top three most frequently identified patterns. Conclusions: The prevalence of biofilm encoding genes, which are associated with multidrug resistance in S. aureus strains isolated from burn patients, is high. Therefore, identification of epidemiology, molecular characteristics, and biofilm management of S. aureus infection in burn units would be helpful.