Background <p><i>Beauveria bassiana</i> is one of the most renowned entomopathogenic fungi and has recently been recognized as a potential biopesticide for a wide range of insect pests. The secondary metabolites of <i>B. bassiana</i> are believed to contribute to its pesticidal activity. Meanwhile, the Egyptian cotton leafworm, <i>Spodoptera littoralis</i>, is a significant pest that causes considerable damage to various key crops.</p> Results <p>In this study, secondary metabolites were extracted using ethyl acetate from both the culture medium filtrate and the mycelia separately. Subsequently, the extracts were separated via thin-layer chromatography into six fractions from the medium filtrate (F0:F5) and ten fractions from the mycelia (M1:M10). All fractions were assessed for their insecticidal effects on second instar larvae of <i>S. littoralis</i>. The treatment of F0 resulted in the highest larval mortality rate of 61.11%, followed by M3 with a mortality rate of 45.19%. M2 and M8 had similar effects, each resulting in a mortality rate of 40.00%. Furthermore, scanning electron microscopy revealed cuticle disorders and deformations in larvae treated with all treatments. Fractions exhibited the highest mortality rates in <i>S. littoralis</i> larvae, and the total injuries were analyzed using GC/MS, which revealed the presence of eight bioactive compounds. Notably, this study represents the first detection of 2,4-di-tert-butylphenol in the mycelia of <i>B. bassiana</i>, a compound known for its high toxicity. In addition, other compounds contributed to the observed experimental results.</p> Conclusion <p>These findings indicate that <i>B. bassiana</i> could serve as a sustainable source of bioactive metabolites with significant insecticidal effects on <i>S. littoralis</i>. This suggests a strong potential for improving integrated pest management (IPM), particularly when large-scale production is implemented.</p> Graphical abstract <p></p>