Virulence of two Iranian isolates of an entomopathogenic fungus, Beauveria bassiana, was evaluated against the tea mealybug, Pseudococcus viburni, by topical exposure and determination of enzymatic activity/expression. The values of 2 × 10³ and 18 × 10⁵ spore/ml were obtained as the LC₅₀ concentrations of BB₃ and Am-118 isolates against third nymphal instars while the LT50 values of the isolates were found to be 3.66 and 6.63 days, respectively. The activities of endo- and exochitanases were assayed in the isolates of B. bassiana that showed higher activity of endochitinase in AM-118 while exochitinase had higher activity in BB₃. Interestingly, the two main proteases Pr1 and Pr2 showed higher activity in AM-118 and BB₃, respectively. Moreover, preparation of a wettable powder formulation based on spores of BB₃ revealed a decrease in viability of spores after 30 days at 30 °C while the formulation showed a significant mortality against nymphs of P. viburni at all intervals after exposure, with the highest value on days 3 and 4. These findings highlighted virulence differences in native isolates of B. bassiana against P. viburni based on results of bioassay and enzymatic virulent factors.