The two spotted spider mite (TSSM) Tetranychus urticae is one of the most economically key mite pests of fields and greenhouse crops over the world. In order to clarify the control efficacy of Neoseiulus barkeri on Tetranychus urticae in the presence of entomopathogenic fungus (EPF) Acremonium hansfodii. This study was conducted to evaluate the control of combining N. barkeri with A. hansfodii against T. urticae. The results showed that the functional response of N. barkeri treated with Ahy1 strain to T. urticae was Holling type II equation. Among these, the highest predation number and the shortest attack time were obtained for the predator feeding on eggs, then followed by feeding on nymphs, female adults ranked as the lowest. That of N. barkeri to T. urticae treated with Ahy1 strain was also Holling type II equation. The predation capacity of N. barkeri on nymphs and female adults of T. urticae treated by Ahy1 strain increased with the increase of the concentration of Ahy1 strain, but that when feeding eggs had no significant difference. Therefore, the combination of EPF with N. barkeri on control of TSSM had synergistic effects, which will provide the guidance to carry out combined utilization of entomogenous fungi with predatory mites.