Crown gall of grapevine (Vitis spp.) is a serious and economically important disease caused by the bacterial pathogen Rhizobium vitis, which transforms healthy plant cell genomes leading to hypertrophic and hyperplastic growth of affected plant cells. Recent studies have documented a strong inhibitory effect against Japanese tumorigenic R. vitis isolates by a newly identified non-tumorigenic strain of R. vitis, ARK-1. We conducted co-inoculation assays in tomato (Solanum lycopersicum) and wine grape cultivars (Vitis vinifera) with four tumorigenic isolates of R. vitis from Virginia. These tumorigenic isolates were co-inoculated with ARK-1 in various ratios and resulting gall incidence and gall size were measured. Analysis was conducted with the generalized linear mixed model (GLIMMIX) in SAS (ver. 9.4). ARK-1 significantly reduced both the mean probability of gall formation and the mean gall size (P < 0.05). ARK-1 efficacy against combinations of two or four tumorigenic isolates and up to twice as many cells of tumorigenic isolates was also significant. However, there was an indication of a loss of efficacy when ARK-1 was challenged with four isolates at four times the cell number of ARK-1. Also, the efficacy of ARK-1 was influenced by both the specific isolate and host plant used in the study. Our results suggest that ARK-1 has promising potential as an effective biological control agent for grapevine crown gall in the United States.