This study was conducted to evaluate the toxic and biochemical effects of multiple applications with Biofly(Beauveria bassiana) and SpinTor^® (spinosad)on nurse and field honey bee (Apis mellifera L.) workers under laboratory conditions. The mortality percentages of workers treated with both biopesticides were determined after 24 h of one, two and three daily applications. The lethal concentrations of spinosad that caused 50% mortality (LC₅₀) were estimated. The effects of two biopesticides on the acetylcholinesterase (AChE) activities in heads, thoraces, and abdomens of surviving nurse and field workers were determined in vivo after 24 of single, two and three daily applications. The results indicated that spinosad was higher toxic than Biofly to both nurses and field worker. Also, the mortality percentages after 24 h of three daily applications of Biofly and spinosad were higher than that of two daily applications whatever worker type. In the same pattern, the mortality percentages after two daily applications were higher than that of single application. On the other hand, Biofly and spinosad were more toxic to foragers than to nurses after 24 h of one application, or two or three daily applications. Furthermore, our findings indicated that mean values of AChE activity in heads of nurse and field workers fed sugar syrup with 0, 187.5, 375, 750, 1500 and 3000 mg L^-1 of Biofly, and 0, 2.5, 5, 10 and 20 mg L^-1 of spinosad were higher than those in both thoraces and abdomens after 24 h of all treatments. Also, the average of AChE activities in heads was significantly (p > 0.05) higher than that obtained in both thoraces and abdomens whatever tested biopesticide, worker type or number of applications. In addition, tested concentrations caused various degrees of inhibition and activation in AChE activity in heads, thoraces and abdomens when compared with controls. Summed data revealed that Biofly and spinosad increased AChE activity of both worker types after 24 h of applications however it repeated or not.