Abstract: Fresh walnuts have short shelf life due to they are prone to mildew, which greatly limits their circulation distance and economic benefits. In this paper, the effect of chlorine dioxide (ClO₂) on the control of mildew was studied by isolation and reinfection of pathogens, morphological and molecular identification, in vitro and in vivo antibacterial tests, and quality evaluation of fresh walnuts after treatment using 'Qingxiang' walnuts as test materials. The results showed that there were two main pathogens isolated from the mildew zone of fresh walnuts, Penicillium sp and Aspergillus tabinus, and two main pathogens isolated from the sticky zone of fresh walnuts, Pseudomonas eucalyptus and Micrococcus flavus. In vitro, fumigation with 0~40 mg/L ClO₂ had a significant dose-effect relationship on the inhibition of these four pathogens. The IC₅₀ value of the action was between 7 and 20 mg/L. ClO₂ of 16, 30 and 40 mg/L inhibited the propagation and growth of Penicillium sp, Aspergillus tabinus and two kinds of bacteria in vitro by 100%. The antibacterial role of slow-release ClO₂ fumigant presented by inhibiting both the germination of mold spores and mycelium growth. 40 mg/L ClO₂ fumigation significantly (P<0.05) reduced the fungal and bacterial colonies of fresh walnuts during shelf life at 25 and 5 ℃, maintaining better sensory quality, and extending the consumer acceptance period of fresh walnuts to 12 days at 25 ℃ and 48 days at 5 ℃. The combined use of walnut green peel extract (GE) had no significant improvement on the ClO₂ preservation effect. 40 mg/L ClO₂ fumigation has been identified as a convenient and efficient shelf life preservation technology for fresh walnuts.