Organic matter, temperature and pH were studied on bactericidal efficacy of chlorocresol nanoemulsion disinfectant. Suspension quantitative killing test method was used with E. coli as test bacteria. The results showed that 0%, 25% and 50% calf serum had no effect on the bactericidal efficacy of chlorocresol nanoemulsion disinfectant. The bactericidal efficacy of the disinfectant increased with the prolongation of action time, both（10±1） ℃ and（20±1） ℃ had serious effects, but（30±1） ℃ had no effect. pH 4.64, 6.67 and 8.68 had all no effect. These results indicated that organic matter and pH had no effect on the bactericidal action of chlorocresol nanoemulsion disinfectant against E. coli. However, different temperatures had different effects, so attention should be paid to in clinical application.