The essential oil from Teucrium polium was evaluated for its adverse effects on larval instars of Musca domestica. The essential oil was blended with a diet at a concentration that produced 50% mortality of subjected insects (LC50) (80 ppm). To learn about the situation of digestive enzymes of M. domestica treated with the essential oil, third larval instars were dissected under non-active enzyme conditions and their midguts were removed. The supernatant was used as an enzyme source after homogenization and centrifugation of the homogenates. Results revealed that some main digestive enzymes in the larval midgut were adversely affected when exposed to the food-incorporated essential oil. Proteinase extracted from larval midgut hydrolyzed the synthetic substrates B-Arg-pNA, Z-Arg-Arg-PNA and Z-Phe-Arg-PNA for trypsin, cathepsin B and cathepsin L activities, respectively, in control and treated larvae. The essential oil caused a reduction of 61.5% in tryptic activity. Significant 69% and 79% reductions were also observed in cathepsin L and B activities, respectively. Carbohydrase activities of α-amylase, α-glucosidase and β-glucosidase were detected in larval midgut extract. All assayed carbohydrases were affected by the essential oil. The most notable impact, a 93% reduction, was observed in α-amylase. Decreases of 69.5% and 42% were obtained in α-glucosidase and β-glucosidase activity, respectively. This study indicated that the larvicidal effect of the essential oil from Teucrium polium may be due to its detrimental effects on digestive enzymes. It seems that the detrimental effect of the oil can be due to both the inhibitory nature of the oil and the destruction of the midgut epithelium.