Head lice infestation is an emerging social problem in undeveloped and developed countries. Because of louse resistance increasing, several long-used insecticidal compounds have lost their efficacy, and alternatives, such as essential oils, have been proposed to treat this parasitic infestation. The present study investigated the efficacy of two natural substances: tea tree (Melaleuca alternifolia) oil and nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) against lice and its eggs. Products were used alone and in combination (ratio 1:1 and 1:2) from 8 % dilution. The in vitro effect of natural substances at different concentrations were evaluated against 69 head lice (adults and nymphs) and 187 louse eggs collected from school children in Chieti-Pescara (Central Italy) over a 6-month period. The lice mortality was evaluated for 24 h by a stereo light microscope. The ovicidal activity was monitored by microscopic inspections for 15 days. Tea tree oil was more effective than nerolidol against head lice with 100 % mortality at 30 min and 1 % concentration. On the contrary, nerolidol expressed a more pronounced ovicidal activity inducing the failure of 50 % of the eggs to hatch at 1 % concentration after 4 days; the same effect was achieved by using a twice concentration of tea tree oil. The association of the two substances both in ratios 1:1 and 1:2 combined efficaciously their insecticidal and ovicidal effect; in particular, the ratio 1:2 (tea tree oil 0.5 % plus nerolidol 1 %) acted producing both the death of all head lice at 30 min and the abortive effect of louse eggs after 5 days. These results offer new potential application of natural compounds and display a promising scenario in the treatment of pediculosis resistant cases. The development of novel pediculicides containing essential oils could be, in fact, an important tool to control the parasitic infestation.