Wood vinegar is a by-product of the pyrolysis of organic raw materials. In this study, we investigated the chemical composition and biological activity of industrial wood vinegar derived from the pyrolysis of wood pruning waste. The composition of wood vinegar was characterized using liquid chromatography (LC) and gas chromatography–mass spectrometry (GC-MS). Wood vinegar bioactivity was tested against Bactrocera oleae under field conditions in an olive grove for two years. Furthermore, wood vinegar was applied in a greenhouse experiment with strawberry plants and in a strawberry field infested with the nematode Meloidogyne incognita. Finally, a seed root length bioassay was performed to evaluate the phytotoxicity or biostimulation of wood vinegar on Eruca sativa, Lactuca sativa, Lens culinaryis, Lolium multiflorum, and Solanum lycopersicum. Our results showed that wood vinegar had a pH of 3.2, with high concentrations of acetic acid (27,840.16 mg L−1) and phenols (54.00 mg L−1). No repellent effect against B. oleae was observed when wood vinegar was applied as an aerosol in olive groves. On strawberry plants in greenhouse conditions, wood vinegar showed phytotoxic effects at high concentrations, resulting in a decrease in the total yield of the plants. In the field, at a 1% concentration, wood vinegar led to a significant 15% reduction in the infection caused by M. incognita in strawberry plants. Finally, in the in vitro crop bioassay, wood vinegar demonstrated remarkable phytotoxicity effects at high concentrations while promoting root growth when diluted. The efficacy of wood vinegar displayed considerable variability based on concentration and delivery system, emphasizing the need for careful evaluation when considering its application, particularly in diverse crops and production systems.