Abstract-Poly-hexamethylene biguanide hydrochloride (PHMB) and 1-bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione (BCDMH) are widely used for surface disinfection. Here, we examined the bactericidal effects of the mixture of PHMB and BCDMH (named PB) combined in different ratios on S. aureus, and studied effects of temperature, pH and bovine serum albumin (BSA , as organic interferent) on PB bactericidal activity with suspension quantitative germicidal method. We also observed surface structure changes and ATP level in S. aureus treated with PB with scanning electron microscope and multi-function microplate reader, respectively. The results showed that mixture of 10 mg/L PHMB and 10 mg/L BCDMH could meet the bactericidal requirements according to Technical Standard for Disinfection (China 2002), producing similar bactericidal activity to those exhibited by 320 mg/L PHMB and 60 mg/L BCDMH, separately. The results showed that PB was competent to kill bacteria quickly and bactericidal activity increased with time within 12 min. Temperature had no significant effect on PB bactericidal activity. PB offered satisfied bactericidal activity within 3.0 to 5.0 pH range. BSA significantly reduced bactericidal activity of PB. In addition, PB treatment could result in protuberance on S. aureus surface, and bring about a rapid decline in intracellular ATP level.