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Ethanol solutions of five fractions obtained from essential oil of sweet basil Ocimum basilicum L. (Lamiales: Lamiaceae) (F1–F5) were tested for their antifeedant properties against 2nd instar gypsy moth larvae, Lymantria dispar L. (Lepidoptera: Lymantriidae), in laboratory non-choice and feeding-choice experiments. Prior to bioassays, the chemical composition of each fraction was determined by gas chromatography analyses. Significant larval deterrence from feeding was achieved by application of tested solutions to fresh leaves of the host plant. The most effective were were F1 (0.5%), F4 (0.05, 0.1, and 0.5%), and F5 (0.1 and 0.5%), which provided an antifeedant index > 80% after five days. A low rate of larval mortality was observed in no-choice bioassay. In situ screening of chlorophyll fluorescence as an indicator of plant stress level (assessed by the induced fluorometry) confirmed that the tested compounds did not cause alternations in the photosynthetic efficiency of treated leaves.