Abstract Supercritical fluid extraction combined with two fractionation methods, sorption and series of separators, was applied on Eucalyptus globulus leaves in order to maximize the insecticidal activity of extracts against Leptinotarasa decemlineata larvae. The yields and composition of eucalyptus extracts and essential oil were compared. The major compounds in the essential oil isolated with the yield 26.7 mg/g were 1,8-cineole (42.6 wt%), α-pinene (10.7 wt%) and globulol (5.9 wt%). The concentration of total volatiles in the extracts varied from 2.8 to 59.5 wt%, depending on the extraction and fractionation conditions. The extract obtained at 50 °C and 9 MPa exhibited the highest acute toxicity with LD50 = 38 μg. ScCO2 extracts exhibited several times stronger insecticidal activity in terms of chronic toxicity, antifeedant activity and growth inhibition than essential oil. Increase in the concentration of volatiles in isolates resulted into increase of chronic toxicity and antifeedant activity while growth inhibition decreased.