The cotton leaf worm, Spodoptera littoralis, is one of the most injurious agricultural pests in Egypt. In the present study, the interaction between spinosad and Bacillus thuringiensis, using different pairings, in third and fifth instar larvae was determined. Activities of α- and β-non-specific esterases and acetylcholinesterase (AChE) were also determined. Additionally, AChE transcript abundance using the real-time polymerase chain reaction (PCR) was detected. The cumulative percentage mortality of S. littoralis, third and fifth instar larvae, to spinosad and B. thuringiensis were increased gradually by increasing the concentrations. Spinosad was more potent than B. thuringiensis. Sequential application of these bio-insecticides, on the LC₂₅ level on third and fifth instar larvae, when spinosad was first applied, showed synergistic interaction and additive effect, respectively. On the other hand, when B. thuringiensis was first applied, using the same timing of application, additive interaction was observed for both third and fifth instars. Individual, simultaneous and sequential applications of these bio-insecticides induced variable changes in α- and β-esterases and AChE activities. Real-time PCR (qRT-PCR) revealed an increase in transcript abundance up to 2.0-fold in third instar larvae in case of sequential and simultaneous applications. Based on the results herein, integrated pest management programme using spinosad and B. thuringiensis can be successfully developed for the control of S. littoralis.