Abstract: Background and Objective: Methicillin-Resistant Staphylococcus aureus (MRSA), is a common antibiotic-resistant bacterium leading to severe community-acquired infections with limited treatment options. The present study was designed to analyze the antibacterial activity of hydroalcoholic extract of Carthamus tinctorius L., against Methicillin-Resistant Staphylococcus aureus (MRSA). Materials and methods: The evaluation of Carthamus tinctorius L. was done by agar well diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Further, the production of virulence factors like adhesion, biofilm formation, hemolysis, staphyloxanthin production and related genes were also investigated to confirm antibiofilm activity. Results: In the agar well diffusion method, the zone of inhibition was observed at low dosage (1.6 mg mL–1) and increased with higher dosage. In, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), 50% inhibition of Methicillin-Resistant Staphylococcus aureus (MRSA) were observed in 25 mg mL–1 and 100% was observed in 50 mg mL–1 which showed decreased biofilm formation, hemolytic activity, staphyloxanthin production and its Hydrogen Peroxide resistance. Modulation of mecA and spA genes was observed on treatment with extract by decreasing its gene expression. Conclusion: Since, hydroalcoholic extract act against the virulence factor involved in biofilm formation, it can be used as an alternative substitute for antibiotic treatment.