Inactivation of Gram-negative Escherichia coli and Gram-positive Staphyloccocus aureus in litchi juice by DMDC combined with nisin was individually investigated. A 1.66 log cycles reduction of E. coli and 2.03 log cycles reduction of S. aureus in litchi juice (pH 4.5) added without nisin was achieved as exposed to 150 mg/l DMDC at 30 °C for 1 h, and the inactivation rate of E. coli and S. aureus during initial 1 h was far greater than during the remaining 5 h. As exposed to 150 mg/l DMDC at 30 °C for 1 h, the inactivation of E. coli and S. aureus in the litchi juice showed a trend toward increase with increasing of nisin addition level in the range from 0 to 200 IU/ml. Moreover, DMDC and nisin exhibited a synergistic effect on the inactivation of E. coli and S. aureus in litchi juice, and the inactivation of E. coli and S. aureus in the litchi juice also depends on the temperature of litchi juice, pH value of litchi juice and DMDC concentration when treated with DMDC and nisin. In addition, E. coli showed higher resistance to nisin as comparing with S. aureus. After E. coli and S. aureus in the litchi juice of pH 4.0 were individually treated with 150 mg/l DMDC combined with 200 IU/ml nisin at 30 °C for 1 h, a complete inactivation of S. aureus (6.59 log cycles) was achieved, but only 3.52 log cycles reduction of E. coli was observed.