In clinical practice, the important hygienic prevention of bacterial pathogen spread is disinfection of potentially contaminated area. Benzalkonium bromide and chlorhexidine acetate are commonly used disinfectants with a broad spectrum of antimicrobial effect. It is vital to inhibit the spread of pathogen in hospital. However, a large number of pathogens with the decreased antiseptic susceptibility have been isolated from clinical samples which showed an increased minimal inhibitory concentration (MIC) against those antiseptics. These resistant pathogens are the major causes for nosocomial cross-infections in hospital. The present study demonstrated the utility of Oxford plate assay system in determining the potential disinfectant resistance of bacteria. The microbiological assay is based on the inhibitory effect of tested disinfectants upon the strains of Staphylococcus aureus and Escherichia coli. Statistical analysis of the bioassay results indicated the linear correlation (r = 0.87-0.99, P < 0.01) between the diameter of growth inhibition zone and the log dosage of the tested disinfectants. Moreover, comparison of inhibitory efficacy of benzalkonium bromide upon 29 S. aureus strains isolated from clinical samples by both Oxford plate method and broth dilution method showed that the diameter of growth inhibition zone has significantly negative correlation with the minimal inhibitory concentration (MIC) (r = -0.574, P < 0.001). These results suggest that the Oxford plate is a simple and time-saving method in detecting potential clinical disinfectant resistance and its usefulness for routine surveillance of pathogenic resistance to disinfectants warrants further investigation.