Objective To develop an optimized BZK gel with good pharmaceutical effect and less toxicity to vaginal mucosa. Methods Four methods as below were used to research the spermicidal activity of BZK gel: 1) in vitro spermicidal test; 2) in vivo spermicidal test in rabbits; 3) anti-fertility test in rabbits; 4) contraceptive test in rabbits. Mucosal irritation test was used in rats to evaluate the safety of optimized BZK gel. Microbiological assessments were used to research anti-STI pathogens (including treponema pallidum, neisseria gonorrhoeae, trichomona vaginalis, candida albicans, ureaplama urealyticum, herpes simplex virus type-2, chlamydiae trachomatis) effect of optimized BZK gel. Results In vitro spermicidal test, EC50 of BZK gel was 0.029mg/ml, a little higher than that of N-9 (0.019mg/ml). The MIC of BZK gel was 0.25mg/ml, similar to that of N-9 (0.20mg/ml). The vaginal mucosal irritation test in rats showed that 0.429% BZK gel showed only minimal vaginal irritation, and did not damage the vaginal epithelium or cause local inflammation in rats. Microbiological assessments showed that optimized BZK gel could inhibit or inactivate common STI pathogens even after 3-fold or 5-fold dilution. Conclusion Optimized BZK gel was an effective microbicides. Objective To develop an optimized BZK gel with good pharmaceutical effect and less toxicity to vaginal mucosa. Methods Four methods as below were used to research the spermicidal activity of BZK gel: 1) in vitro spermicidal test; 2) in vivo spermicidal test in rabbits; 3) anti-fertility test in rabbits; 4) contraceptive test in rabbits. Mucosal irritation test was used in rats to evaluate the safety of optimized BZK gel. Microbiological assessments were used to research anti-STI pathogens (including treponema pallidum, neisseria gonorrhoeae, Effect of optimized BZK gel. Results In vitro spermicidal test, EC50 of BZK gel was 0.029 mg / ml, a little higher than that of N-9 The MIC of BZK gel was 0.25 mg / ml, similar to that of N-9 (0.20 mg / ml). The vaginal mucosal irritation test in rats showed that 0.429% BZK gel showed only minimal vaginal irritation , and did not damage the vaginal epithelium or cause local inflammation in rats. Microbiological assessments showed that optimized BZK gel could inhibit or inactivate common STI pathogens even after 3-fold or 5-fold dilution. Conclusion Optimized BZK gel was an effective microbicides.