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The potential interaction of fluctuating temperature and the virulence of entomopathogens has implications for biological control. The objective of this study was to investigate the effect of entomopathogenic fungi (Metarhizium brunneum, Beauveria bassiana) on noctuid pest caterpillars (Spodoptera littoralis, Heliothis virescens) under constant and fluctuating temperature regimes. The results revealed similar virulence of entomopathogenic fungi under fluctuating temperature (20–30 °C or 15–35 °C) compared with constant temperature (25 °C). Experiments with cotton leaves as food confirmed that S. littoralis was less susceptible to entomopathogenic fungi (M. brunneum) than H. virescens. Results of additional experiments with H. virescens larvae, B. bassiana, and artificial diet were comparable to experiments with M. brunneum and cotton leaves, despite that susceptibility to B. bassiana was three orders of magnitude lower than to M. brunneum. The fact that both fungus species showed reduced growth on medium under fluctuating temperatures when compared to constant temperatures did not translate to an interaction of temperature regime and the virulence against noctuid larvae. Our study implies that virulence studies with noctuid larvae under constant temperatures using plant material or artificial diet might be fair models also for environments with fluctuating temperatures.