The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and β-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5–25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 ± 1.8% over controls. The essential oils alone reduced GST activity by 3–20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58–76% and reduced GST activity by 3–85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti.