Aims: Benzalkonium chloride is used to disinfect hospital instruments to prevent nosocomial infection caused by microorganisms, such as Methicillin Resistant Staphylococcus aureus (MRSA). There are strains of MRSA isolated from hospitals that were found to be resistant towards benzalkonium chloride. This research was aimed to compare the affectivity of different concentrations of benzalkonium chloride to inhibit the growth of Hospital-Associated MRSA (HA-MRSA) and determine the Minimum Inhibitory Concentration (MIC) of benzalkonium chloride against HA-MRSA. Methodology and results: The samples were five HA-MRSA isolates obtained from Dr. Soetomo Hospital Surabaya. It was identified by amplification of SCCmec genes. The HA-MRSA with SCCmec type III was divided into six flasks based on the concentration of benzalkonium chloride in their inoculation media (0 μg/mL, 0.625 μg/mL, 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, and 10 μg/mL). The growth of HA-MRSA in media was determined by the number of colonies after treatment. The result showed that the MIC of benzalkonium chloride for HA-MRSA was 5 μg/mL, where no growth of bacterial was observed. There was significant difference in MRSA colony count between different groups of benzalkonium chloride concentrations (p = 0.001), and there was negative correlation between benzalkonium chloride concentration and HA-MRSA growth (p = 0.0001 and r = -0.880). Conclusion, significance and impact of study The concentration of benzalkonium chloride influences the growth of HA-MRSA. The higher the concentration, the fewer HA-MRSA growth. Application of benzalkonium chloride according to MIC will prevent HA-MRSA resistance towards benzalkonium chloride.