The virulence and proteolytic activity of some entomopathogenic fungi isolates, viz., Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, and Trichoderma harzianum, against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), were evaluated. Common maize plants (Zea mays L.) infested with females of T. urticae were treated in vivo by spraying with suspensions of 1 × 10 8 conidia ml −1 concentration of selected isolates. Lethal effects of fungal isolates were assessed as percentages of daily mortalities of mites, compared to the mortality in control. Virulence of the fungi isolates was estimated based on the LC 50 values calculated by probit analysis for the individuals treated by 1 × 10 5 conidia ml −1 concentration. Proteolytic activity of isolates was assayed on casein substrate to reflect their virulence towards T. urticae. The mite mortality rates increased with increasing conidial concentrations as well as days after treatment. The mortality rate caused by M. anisopliae isolate varied from 18.75 to 85%, with LC 50 value of 4.6 × 10 5 conidia/ml and LC 90 value of 2.4 × 10 8 conidia/ml during 7 days, respectively. The isolate of B. bassiana caused 15 to 70% mortality, and its LC 50 and LC 90 values estimated 3.3 × 10 6 and 7.8 × 10 9 conidia/ml, respectively. However, V. lecanii isolate caused 11.25 to 72.50% mortality with LC 50 of 5.2 × 10 6 conidia/ml, while T. harzianum was potentially less virulent than other isolates causing 8.75 to 63.75% mortality rate to T. urticae with LC 50 of 9.4 × 10 6 conidia/ml. M. anisopliae showed the highest proteolytic activity at all concentrations, followed by B. bassiana in 3rd, 5th, and 7th day post treatment. These findings recommend the selection of virulent fungal isolates for use as natural and environmentally safe agents in biological control programs to combat mite pests.