Despite the fact that temperatures fluctuate on a daily basis in the field, the effect of fluctuating temperature on the susceptibility of pest insects to agricultural management tactics are lacking. We thus tested how the development of Spodoptera littoralis and Heliothis virescens (Lepidoptera: Noctuidae) is influenced by exposure to an insecticidal genetically engineered pulverized powder of, Bollgard II cotton producing the Bt proteins Cry1Ac and Cry2Ab, or an entomopathogenic fungus (Beauveria bassiana) under fluctuating temperatures in comparison with constant temperature. When temperature fluctuated symmetrically around 25 °C either in small range (± 5 °C) or in a large range (± 10 °C) on daily basis, development of both lepidopteran species was positively or not influenced in absence of additional stressors. Both species were affected by Bt-cotton when added at low concentrations to the artificial diet, but their reaction to Bt-cotton did not differ between fluctuating temperatures compared to a constant 25 °C. When H. virescens larvae were dipped into spore suspension of B. bassiana, all larvae reared at constant 25 °C died before pupation, while no detrimental effect of the fungus was visible in the fluctuating temperature regimes. In contrast, S. littoralis was not affected by the fungus in any treatment. Since fungal growth was reduced at fluctuating temperatures, this might have affected the infection rate in H. virescens. The results of this study indicate that fluctuating, compared to constant temperatures, can influence the susceptibility of pest insects to biological control agents, while temperature regimes seem to be less important for the efficacy of insecticidal genetically engineered crops.