Chromosome architecture plays a crucial role in bacterial adaptation, yet its direct impact remains unclear. Different bacterial species and even strains within the same species exhibit diverse chromosomal configurations, including a single circular or linear chromosome, two circular chromosomes, or a circular-linear combination. To investigate how these architectures shape bacterial behavior, we generated near-isogenic strains representing each configuration in Agrobacterium tumefaciens C58, an important soil bacterium widely used for plant genetic transformation. Strains with a single-chromosome architecture, whether linear or circular, exhibited faster growth, enhanced stress tolerance, and greater interstrain competitiveness. In contrast, bipartite chromosome strains showed higher virulence gene expression and enhanced transient plant transformation efficiency, suggesting a pathogenic adaptation. Whole-transcriptome analysis revealed architecture-dependent gene expression patterns, underscoring the profound impact of chromosome organization on Agrobacterium fitness and virulence. These findings highlight how chromosome structure influences bacterial adaptation and shapes evolutionary trajectories.