Biofly (Beauveria bassiana) and SpinTor® (Spinosad) are increasingly applied as biopesticides throughout Egypt to control various agricultural pests. We investigated, in a previous study, the acute toxicities of the two biopesticides among others and their effect on acetylcholinesterase (AChE) activity of honey bee (Apis mellifera L.) workers. In this study, we are focusing on the acute toxicities and the potential side effects of multiple applications with biofly (Beauveria bassiana) and SpinTor® (Spinosad) biopesticides on AChE activity of larvae of honey bee workers. Chosen groups of young worker larvae, in a brood comb of honey bee colony, were fed once, twice or three times at 1-day intervals on sugar syrup 1:1 (w/v) containing different concentrations of the two biopesticides. The mortality percentages of treated worker larvae were determined after 24 h of one application, or two or three daily applications, and the lethal concentrations that caused 50% mortality (LC₅₀) were estimated to determine the acute toxicity of Biofly and Spinosad to worker larvae. Also, the impacts on AChE activity of larvae were determined in vivo after 24 and 96 h of single application or two daily applications. According to the LC₅₀ values, Spinosad showed higher toxic actions to worker larvae comparable to Biofly. Also, the acute toxicity (after 24 h) of three daily applications of Biofly (1905 mg L^-1) or Spinosad (12.04 mg L^-1) was higher than the corresponding value of two daily applications (3847 and 21.45 mg L^-1, respectively).The same trend, the acute toxicity of two daily applications was higher than that of single application (5113 and 51.29 mg L^-1, respectively). Therefore, there were lethal cumulative effects of Biofly and Spinosad on worker larvae. Furthermore, our findings indicated that the average of AChE activities in larvae fed twice on sugar syrup with Biofly or Spinosad was significantly (p > 0.05) higher than that in larvae fed once after 24 and 96 h. Also, Biofly when found in sugar syrup at tested concentrations has activator effects after 24 h of application, and inhibitory effects after 96 h of application on AChE activity in worker larvae fed once or twice. In addition, Spinosad showed activator effect only after 24 h of single application, and inhibitory effects after 24 h of two daily applications and after 96 h of one or two daily applications on AChE activity in worker larvae.