The water buffalo farm with intensive breeding techniques promotes the spread of lice (Haematopinus tuberculatus), leading to a reduction in meat and milk productions. Current control measures include the use of chemicals, which in the long-term lead to the development of resistance. Given the lack of alternative solutions, the aim of this study was to evaluate the repellent effect of a low impact by-product, the wood vinegar (WV), against H. tuberculatus using a new in vitro bioassay test. The test consisted of a glass Petri dishes subdivided in 3 separate areas. WV was tested at different concentrations (100%, 75%, 50%, 25%). Each of the 3 replicates was performed with 10 adults, at 27 °C and 75% relative humidity. Observations were made 5, 15, 30, 60, 90, 120 and 180 min after application to evaluate movement, the living/dead ratio and the ability to overcome the obstacle area of the lice, in terms of comparative avoidance (%). Additional in vitro cytotoxicity tests were performed. The test performed showed a repellent effect of 77% for the 100% WV concentration against lice of both sexes 30 min after exposure, similar (P < 0.05) to the repellent effect of deltamethrin (77%). The in vitro bioassay developed during the experiment proved to be reliable and can be used to evaluate the repellent effect of new molecules against lice before testing them in vivo. Furthermore, WV has a potential to be a valid tool for the control of buffalo pediculosis, although in vivo studies have to be undertaken.