This research investigated the Eucalyptus cinerea leaves efficiency in the Agrobacterium tumefaciens biocontrol, the causative agent of crown gall. GC-MS analysis of the essential oil (EO) showed that the main components were 1,8-cineole (61%) and camphene (15.13%). Thanks to its polyphenols, flavonoids, quinones, terpenoids, alkaloids, and tannins richness, the EtOAc-F exhibited the most potent antibacterial activity in vitro. Indeed, compared to the other fractions, it has the lowest MIC and MBC values of 0.312 mg/mL and 2.5 mg/mL, respectively. The GC-MS analysis of EtOAc-F confirmed its richness in antibacterial compounds including gallic acid (7.18%), shikimic acid (5.07%), and catechin (3.12%). The time-kill curve assay of EtOAc-F (2.5 mg/mL) showed a potent bactericidal effect after 20 min of direct contact with A. tumefaciens. In planta experiments, gall weights were significantly reduced when EtOAc-F was applied at 0.625 and 2.5 mg/wounds. Besides, the disease reduction rates in gall weight were 95% and 97.5%, respectively. Interestingly, no phytotoxic effect was observed since tomato seeds germinated in the presence of the different concentrations of EtOAc-F. These results suggest that EtOAc-F has a good potential to be a curative biocontrol agent for crown gall disease.