Objective(s):Staphylococcus aureus is a Gram-positive pathogen, well known for its resistance andversatile lifestyle. Under unfavourable conditions, it adapts biofilm mode of growth. For staphylococcal biofilm formation, production of extracellular polymeric substances (EPS) is a pre-requisite, which is regulated by ica operon-encoded enzymes. This study was designed to know the impact of ascorbic acid on biofilm formation and colony spreading processes of S. aureus and MRSA. Materials and Methods: The isolates of methicillin-resistant S. aureus (MRSA) used in present study, were recovered from different food samples. Various selective and differential media were used for identification and confirmation of S. aureus. Agar dilution method was used for determination of oxacillin and ascorbic acid resistance level. MRSA isolates were re-confirmed by E-test and by amplification of mecA gene. Tube methods and Congo-Red agar were used to study biofilm formation processes. Gene expression studies were carried on real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results: The results revealed the presence of mecA gene belonging to SCCmecA type IV along with agr type II in the isolates. In vitro studies showed the sub-inhibitory concentration of oxacillin induced biofilm production. However, addition of sub-inhibitory dose of ascorbic acid was found to inhibit EPS production, biofilm formation and augment colony spreading on soft agar plates. The inhibition of biofilm formation and augmentation of colony spreading observed with ascorbic acid alone or in combination with oxacillin.  Moreover, gene expression studies showed that ascorbic acid increases agr expression and decreases icaA gene expression. Conclusion:The present study concluded thatascorbic acid inhibits biofilm formation, promotes colony spreading and increases agr gene expression in MRSA.