The two-spotted spider mite, Tetranychus urticae, is an important mite pest worldwide. It often leads to reduced crop yields or poor marketability of the produce and has already developed resistance to many acaricides. In this study, the pathogenicity of the entomopathogenic fungus (EPF), Acremonium hansfordii, to T. urticae and its side effects on the predatory mite Neoseiulus barkeri were compared. The toxicity of A.hansfordii was evaluated on T. urticae at 1.0×10⁴, 1.0×10⁶, 1.0×10⁸ conidia/mL concentrations and predatory mite N. barkeri at 1.0×10⁸ conidia/mL under experimental conditions at 25±1°C, 75±5% RH and 16L: 8D photoperiod. The effects of EPF on the development of the F₀ and F₁ generation of predatory mites were also assessed at 1.0×10⁸ conidia/mL concentration. The results showed that the corrected mortality of T. urticae at three tested concentrations were 36.67%, 53.67% and 66.33% within 11d, and the median lethal time were 14.39 d, 9.84d and 7.99d, respectively. The corrected mortality of N.barkeri was only 3.9% after 11d at 1×10⁸ conidia/mL concentration. Some hyphae of A.hansfordii were detected on the surface of treated T. urticae but not on N.barkeri body after 7d. After N.barkeri females were treated with 1.0×10⁸ conidia/mL, the preoviposition period of the F₀ generation was prolonged 1.56 times and the other life history parameters have no significant differences; similar patterns were also shown in the F₁ generation. A. hansfordii showed strong pathogenicity against T.urticae but did not significantly adversely influence N.barkeri. Therefore, the combination of A.hansfordii and N.barkeri has the potential to be used for the control of T. urticae in the future.