The objective of this study was to evaluate the potential synergistic effect of ethanol on a combination of OPA and DDAC against the spores of Bacillus subtilis var.Niger.The quantitative carrier test for sporicidal testing of high-level disinfectants according to the guideline of China (Technical Standard for Disinfection 2002) was used as method.Considerable synergistic effect was observed after a 30 min treatment at 20℃.There was an augment in mean log reduction as the concentration of DDAC was increased ranging from 0.2 to 3 g 1-1 in combination with 6 g 1-1OPA.10 and 20% ethanol in combination with 6 g 1-1 OPA and 2 g 1-1 DDAC caused more than a 3 log reduction while either 6 g 1-1 OPA, 2 g 1-1DDAC and 20% ethanol alone or a combination of two of the three agents produced less than a 1 log reduction.Further, 40 min exposure time of combination of OPA, DDAC and 20% ethanol led to greater than a 5 log reduction of spores and no spore growth was observed following 60 and 90 min exposures.The synergistic sporicidal effect exhibited by ethanol on a combination of OPA and DDAC can be considered to enhance sporicidal activity for using in situations of sterilization, to reduce in-use concentration of OPA used alone, which may minimize its side effect.OPA may be a more satisfactory and the first choice agent to replace glutaraldehyde (GTA) as a high-level disinfectant for medical devices.