Grapevine crown gall causes significant economic damage in vineyards and nurseries worldwide. Infected vines are not curable due to irreversible genomic transformation by the tumorigenic bacterium Rhizobium vitis. Crown gall results in a gradual decline in yield and vine vigor, then the complete collapse of the vine. R. vitis ARK-1, an antagonistic and non-tumorigenic strain, has been shown to inhibit gall formation when equal cell concentrations of antagonist and tumorigenic strains are co-inoculated in planta. In this study, we tested the efficacy of ARK-1 against higher cell numbers of tumorigenic isolates and evaluated timing and methods of ARK-1 application in in-planta assays in grapevine (Vitis vinifera 'Chardonnay', 'Cabernet Sauvignon', 'Merlot') and tomato (Solanum lycopersicum 'Beefsteak'). ARK-1 significantly (P ≤ 0.01) reduced gall incidence and gall diameter against four times higher tumorigenic bacterial cell numbers. Inoculation of ARK-1 up to 48 hours before or within 6 hours after inoculation with tumorigenic isolates significantly (P ≤ 0.01) reduced gall incidence and gall diameter. Root dipping of grapevine for 24 hours and tomato for 1 hour in the formulated ARK-1 suspension (Kumiai Chemical Industry Co. Ltd., Japan) prior to transplanting in tumorigenic bacteria-infested soil completely inhibited gall formation in the roots. These results are promising and support the development of ARK-1 as a biological control agent to manage grapevine crown gall.