Background Insects withstand foreign substances and infection by expressing robust defense responses, which are mediated by hemocytes, fat body, midgut, and many other tissues. The Egyptian cotton leafworm, Spodoptera littoralis (Boisduval), is a polyphagous pest with considerable economic importance in Egypt and globally. Many control strategies were employed to control this pest. Nowadays, there is a trend to use nanotechnology tools in agricultural practices as they balance minimal concentration and maximum pest control, safe concentration, and reduce the cost of pest control. The present study aimed to evaluate the hematological and histopathological response of S. littoralis larvae post-treatment with sublethal concentrations of emamectin benzoate and its nanoform, besides the silver nanoparticles. Results The results revealed the high toxicity of emamectin benzoate and its nanoform (LC50 values were 0.0524 and 0.023 ppm, respectively). The results also showed that all tested compounds significantly influenced the mean number of laid eggs/female. The emamectin benzoate nanoform (837.3 ± 52.09) was the most efficient compound compared to the control (1999.3 ± 46.5). The hematological responses against the EMB + AgNP were lowered total hemocyte counting (22.41 ± 1.3) compared to the untreated larvae (38.08 ± 0.83). In addition, there were some histopathological changes in the midgut tissues. They were represented as destroying the integrity of the epithelial cells and the ciliated border. The columnar cells began to disintegrate, and the peritrophic membrane became vacuolized. In contrast, the cuticle layers were not affected by various treatments. Conclusion We can conclude that the employment of emamectin benzoate, either in its original form or as its nanoform, is considered a promising substitute for conventional insecticides. The nanoform of emamectin benzoate proved its high efficiency against the larvae of the cotton leafworm, which may allow the application of this formulation at low concentrations.