Staphylococcal biofilms are the prominent cause of chronic wound infection and antibiotic resistance. It acts as a reservoir for bacteria, making wound healing difficult. Biofilm infections increase the hospital stays and cost to the patients. The current study explores the phenotypic and genotypic detection of S. aureus biofilm from chronic traumatic wounds and their association with antibiotic resistance. A prospective observational study was conducted from April 2020 to March 2021. S. aureus isolates were identified by the MALDI-TOF. Antibiotic susceptibility was determined by VITEK 2. Biofilm production detected by tissue culture plate method and associated ica genes were diagnosed through multiplex PCR. Overall, 67 isolates were investigated. The frequency of biofilm production in S. aureus was 73.1%, and most of the isolates were moderate biofilm producers (38.8%). The presence of intracellular adhesion (ica) operon among these isolates was 85.7% and also significantly associated with the strength of biofilm mass formation. Ica A was the predominant biofilm-producing gene (42.9%). Biofilm producing Methicillin-resistant S. aureus were as high as 75%, and multidrug resistant strains were significantly associated with biofilm formation. But frequency of ica genes were noted more in Methicillin sensitive S. aureus (65.2%). High frequency of biofilm in S. aureus of isolates was responsible for the development of chronic non-healing traumatic wounds. Biofilm-producing isolates showed greater multidrug-resistance. Phenotypically MRSA expressed more biofilm, but ica operon was documented in MSSA. It emphasized the further need for ica independent pathway exploration for MRSA biofilm synthesis.