Purpose <p>Antimicrobial resistance poses a significant global health challenge, contributing to a lack of effective therapeutic agents, especially against Gram-negative bacteria. Resistance dissemination is accelerated by horizontal gene transfer (HGT) mechanisms. The extended-spectrum beta lactamases CTX-M confer resistance to several beta-lactams, are usually embedded into plasmids and thought to be mainly disseminated by conjugation. However, an increasing number of isolates carry these enzyme-encoding genes in the chromosome, suggesting that they can spread by other means of HGT. In this study, we aimed to test the involvement of natural transformation in the chromosomal acquisition of a <i>bla</i>_CTX−M gene.</p> Methods <p>Natural transformation assays were performed during motility on wet surfaces. Acquisition of foreign DNA by transformants was screened by antimicrobial susceptibility testing, polymerase-chain reaction (PCR) and whole genome sequencing (WGS).</p> Results <p><i>Acinetobacter baumannii</i> A118, a naturally competent clinical strain, was transformed with naked DNA from <i>Salmonella enterica</i> serovar Typhimurium Sal25, which was isolated from swine meat. The transformation occurred at low frequency (2.7 × 10^− 8 ± 2.04 × 10^− 8 transformants per recipient) and <i>bla</i>_CTX−M was acquired in one transformant, which was named ACI. WGS of the transformant revealed the acquisition of the <i>bla</i>_CTX−M−32 as part of a ca. 36 Kb DNA fragment through an IS<i>Ec9</i>-mediated transposition event; various mobile genetic elements and other resistance genes were co-transferred. The <i>bla</i>_CTX−M−32 gene was subsequently transferred within <i>A. baumannii</i> at a higher frequency (1.8 × 10^− 6 ± 2.49 × 10^− 6 transformants per recipient).</p> Conclusion <p>Our results highlight the importance of natural transformation events in the dissemination of antimicrobial resistance genes and mobile genetic elements between and within species.</p>