Abstract Gram-negative pathogens pose a significant threat due to their propensity for causing various infections, often coupled with formidable resistance to conventional antibiotic treatments. In light of this challenge, the development of antivirulence (AV) compounds emerges as a promising alternative strategy, aiming to disrupt key virulence mechanisms rather than directly targeting bacterial viability. One such compound, aurodox, derived from Streptomyces goldiniensis , has exhibited promising AV properties in our prior studies. Specifically, aurodox caused a marked downregulation in the expression and function of the E. coli type 3 secretion system (T3SS), a needle-like injectosome structure which is deployed to translocate effector proteins from the cytoplasm to the host target cells. However, the broader spectrum of aurodox’s efficacy against T3SS across diverse pathogens remained unanswered, prompting the focus of this work. Using quantitative real-time PCR, we show that aurodox exerts inhibitory effects on selected T3SS in various pathogens, including Salmonella typhimurium, Yersinia pseudotuberculosis , and Vibrio parahaemolyticus . However, aurodox was not a universal blocker of all secretion systems, showing selectivity in its mode-of-action, even within a single strain. This finding was verified using transcriptomics which demonstrated that aurodox selectively blocks the expression of the Salmonella typhimurium SPI-2 type T3SS whilst other pathogenicity islands, including the SPI-1 system were not inhibited. To delve deeper into the mechanisms governing aurodox’s efficacy against these pathogens, we analysed transcriptomic datasets from both E. coli and S. Typhimurium treated with aurodox. By identifying orthologous genes exhibiting differential expression in response to aurodox treatment across these pathogens, our study sheds light on the potential mechanisms underlying the action of this rediscovered antibiotic.