Background <p><i>Acinetobacter baumannii</i> is a major pathogen in hospitals, causing a notable rise in bloodstream infections among inpatients. Its growing resistance to multiple drugs limits treatment options. This study aims to examine the antibacterial effects of gallium nitrate [Ga(NO₃)₃] against <i>A. baumannii</i> and elucidate the underlying molecular mechanism.</p> Methods <p>40 strains of <i>A. baumannii</i> with different antimicrobials susceptibility patterns were isolated from bloodstream infections. The in vitro antibacterial activity of Ga(NO₃)₃ was analyzed by micro-dilution method and time-kill assay. The influence of ferric chloride/hemin on the antibacterial efficacy of Ga(NO₃)₃ was investigated. Transcriptome sequencing was performed to elucidate the antibacterial mechanism of Ga(NO₃)₃. A mouse infection model was conducted to assess its in vivo performance.</p> Results <p>Ga(NO₃)₃ exhibited a potent antibacterial effect in RPMI 1640 medium containing 10% human serum, with MICs ranging from 0.06&#xa0;μg/mL to 0.125&#xa0;μg/mL. The antibacterial activity of Ga(NO₃)₃ was found to be dose- and time- dependent. However, the antibacterial effect of Ga(NO₃)₃ was partially compromised in the presence of exogenous ferric chloride/hemin. Transcriptomics analysis revealed that Ga(NO₃)₃ exerted its antibacterial effect by up-regulating the expression of genes associated with siderophore biosynthesis and transport, while simultaneously disrupting multiple iron-dependent metabolic processes. Ga(NO₃)₃ treatment significantly reduced bacterial load in vivo using a neutropenic mouse thigh infection model.</p> Conclusion <p>This study sheds light on the antibacterial mechanisms of Ga(NO₃)₃ against <i>A. baumannii</i>, suggesting its potential as a promising antibacterial drug for treating bloodstream infections caused by multidrug-resistant <i>A. baumannii</i>.</p>