Abstract: Watermelon fusarium wilt caused by Fusarium oxysporum f. sp. niveum (FON), is a devastating soil-borne disease that seriously damages watermelon production worldwide. Through in vitro and in vivo assays, the inhibition activity of hinokitiol on FON, control efficacy to fusarium wilt of watermelon, and its effect on the physiological and biochemical indexes of FON were determined. The results showed that hinokitiol had an obvious effect against FON, and its EC₅₀ values for inhibition of mycelial growth and spore germination were 31.1 and 45.2 µg/mL, respectively. The pot tests showed that all the three different concentrations (250, 500 and 1000 µg/mL) of hinokitiol had significant inhibitory effects on the fusarium wilt of watermelon. The control efficacy was 75.2% when the concentration of hinokitiol was 1000 µg/mL, which was comparable to that of the control agent carbendazim at a concentration of 500 µg/mL. The tests of physiological and biochemical indicators showed that the relative electrical conductivity of FON increased and the content of fusaric acid significantly declined. The mycelium showed irregular shapes such as distortion, deformity, and entanglement after treatment with hinokitiol. These results suggested that hinokitiol could not only significantly inhibit the growth of FON, but also inhibit the synthesis of toxins and reduce the pathogenicity. Hinokitiol has the potential to be developed as an alternative fungicide for the control of watermelon fusarium wilt. Hinokitiol destroys the cell membrane integrity of the pathogen, but its specific antifungal mechanism still needs further study.