Biofilm formation is a major virulence factor in methicillin-resistant Staphylococcus aureus (MRSA), contributing to chronic infections and antibiotic resistance. The intercellular adhesion icaA and icaB genes are key determinants involved in biofilm synthesis and stabilization. Understanding the prevalence and correlation of these genes with biofilm formation is essential for improved diagnostic and therapeutic strategies. This study aimed to evaluate the prevalence of icaA and icaB genes and their correlation with phenotypic biofilm formation in MRSA isolates from clinical samples. An observational study was conducted over two years in a tertiary care hospital in India. A total of 102 MRSA isolates were phenotypically assessed for biofilm formation using the tissue culture plate method. Genotypic detection of icaA and icaB was performed by real-time PCR. Statistical correlation between gene presence and biofilm formation was analyzed using the chi-square test. Among the 102 MRSA isolates, 36.3% were biofilm producers, including 15.7% strong, 11.8% moderate, and 8.8% weak producers. Genotypic analysis showed that 66.7% of isolates harbored both icaA and icaB, while 20% carried only icaB. A strong correlation was observed between icaA presence and strong biofilm production (p < 0.05), whereas icaB alone did not correlate significantly with biofilm formation. The findings highlight the critical role of icaA in initiating biofilm formation, while icaB may contribute to biofilm stabilization. Comprehensive phenotypic and genotypic screening is essential for understanding MRSA pathogenicity and improving infection control strategies.