Background Plant crown gall disease caused by Agrobacterium tumefaciens causes significant losses in the cultivation of various ornamental and fruit trees. The emission of volatile organic compounds (VOCs) by biocontrol agents (BCAs) has garnered considerable attention due to their notable antagonistic effects. This study evaluated the biocontrol effects of VOCs produced by Pseudomonas chlororaphis subsp. aurantiaca ST-TJ4 against A. tumefaciens PX-1, the causal agent of cherry blossom crown gall.Results The VOCs released by P. chlororaphis subsp. aurantiaca ST-TJ4 significantly inhibited the colony size, cell viability, and swimming motility of A. tumefaciens PX-1, consequently impairing chemotaxis. Moreover, transmission and scanning electron microscopy revealed substantial severe morphological and ultrastructural changes in A. tumefaciens PX-1 cells, accompanied by a significant reduction in their ability to attach to plant roots. Furthermore, VOCs decreased the transcriptional expression levels of virulence-related genes (VirA, VirG, VirD2, VirE3) and three chemotaxis-related genes (CheW1, CheW2, CheA), which play pivotal roles in the pathogenicity of the bacteria. The observed downregulation of the superoxide dismutase (sod) gene indicated oxidative damage to A. tumefaciens PX-1 cells. These gene expression changes explained why A. tumefaciens PX-1 lost its early pathogenicity when inoculated on rose. In the antibacterial substance test, the VOCs of P. chlororaphis subsp. aurantiaca ST-TJ4 exhibited antagonistic effects on A. tumefaciens PX-1, with 2-undecone, 1-nonanol and 2-heptanone identified as the active compounds; among them, 1-nonanol and 2-heptanone exhibited the strongest antibacterial effect.Conclusions The VOCs produced by P. chlororaphis subsp. aurantiaca ST-TJ4 exhibited biocontrol potential against the tree crown gall pathogen A. tumefaciens.