The current investigation was conducted to study the efficacy of natural diatomaceous earth (DE) alone and combined with two fungal pathogens: Metarhizium anisopliae and Beauveria bassiana, as a new formulation against Spodoptera littoralis third instar larvae under laboratory conditions. When M. anisopliae, B. bassiana, and DE were applied alone, the mortalities of larvae were concentration-dependent. The LC₅₀ values were 299.2, 391.5, and 572 ppm, respectively. The combination of DE with M. anisopliae and with B. bassiana enhances the efficacy of both. The LC₅₀ values were 133.77 and 189 ppm, respectively, indicating a toxic potentiation effect. The results demonstrated that the application of DE in a binary combination with fungi significantly reduced the immune enzyme activity (phenoloxidase, prophenoloxidase, lysozyme) and protein concentration compared to the control. These findings are consistent with the theory that diatomaceous earth increases the effectiveness of entomopathogenic fungi against insects by causing cuticle damage, which increases conidial attachment and increases the availability of nutrients for conidial germination. It concluded that adding DE to entomopathogenic fungi helps in improving their quality and efficiency in reducing insect outbreaks, and it suggested participating in sustainable pest management of S. littoralis.