This study aimed to investigate the toxicity of citronella, mustard, and sage essential oils in comparison to their nanoemulsions. Laboratory bioassays were conducted against the 2nd and 4th instar larvae of Spodoptera littoralis. The results indicated that the LC50 values were 0.231, 1.898 and 2.500% for bulk oils of mustard, citronella, and sage against 2 nd instar larvae of S. littoralis, respectively, while they were 0.253, 0.266, and 0.962% for their nanoemulsions, respectively. The LC50 values were 0.889, 3.358 and 0.995% for bulk oils of mustard, citronella, and sage against the fourth instar larvae, respectively, while the LC50 values of their nanoemulsions were 0.657, 0.572 and 0.488%, respectively. The fourth instar larvae were treated with LC50s concentrations of tested essential oils and their nanoemulsions to estimate the biochemical changes. The results showed that there was a significant increase in stimulation of AChE activity, in the case of mustard and citronella bulk and nanoemulsions, while there were no significant changes in sage oil bulk treatment and its nanoemulsion compared with the control. Also, there was a significant decrease in activity of GST enzyme in mustard, citronella bulk oil and mustard nanoemulsion. In contrast, the activity of GST increased significantly in the treatments of sage bulk oil, mustard, citronella and nanoemulsion of sage oil. Alpha esterases activity decreased significantly in all treatments except nanoemulsion of mustard oil which caused a significant increase in the enzyme activities compared with the control.