Three entomopathogenic fungi isolates, i. e., Beauveria bassiana, Paecilomyces fumosoroseus and Metarhizium anisopliae were used as biological control agents against the Egyptain cotton leaf worm, Spodoptera littoralis (Boisd.), which represent the most severe destructive cotton pest in Egypt and many other countries. The entomopathogenic fungi were inoculated YES medium, incubation period was carried out for 19 days at pH 5.4 and 28 – 30ºC. After incubation period, the mats of the fungi were removed and YES broth was mixed individually with chloroform/methanol (2:1, v/v). The mixture was shaken vigorously in a separating funnel and left to settle down forming a dense lower aqueous layer containing the secondary metabolites. Extracted metabolites were then concentrated by using a speed vacuum device (Maxi Dry Plus) to a volume of 1 ml. Virulence of B. bassiana, M. anisopliae and P. fumosoroseus was investigated against 1st instar larvae of the cotton leaf worm S. littoralis. Results obtained revealed that the three metabolites (toxins) were toxic to the 1st instar larvae of S. littoralis. The most effective toxin was M. anisopliae (84.92 %) followed by P. fumosoroseus (66.32%) and B. bassiana (62.65 %). On the other hand, LT50 values in days for M. anisopliae were 8.5414, 6.4808, 4.7721, 3.7211, while for P. fumosoroseus they were 11.0488, 7.7757, 6.4998, 5.1703 and for B. bassiana they were 10.4380, 8.3415, 6.8299, 5.6922 in relation to the concentrations 25, 50, 75, 100% respectively. Treatment of S. littoralis 4th instars with LC50 of the metabolic fungal toxins caused complete disruption of endocuticle of the resulting late 6th instars. Meanwhile, it caused exfoliation of the mid gut epithelium from the underlying circular muscle fibers, leaving a large vacuole or space, disruption of both the peritrophic membrane and columnar cells, beside high vacuolization and collapsing of the fat body.