Abstract <p>The persistent prevalence of multidrug-resistant tuberculosis (TB) and extensively drug-resistant TB has progressively undermined the efficacy of TB infection control measures. Thus, developing innovative drugs that act on novel <i>Mycobacterium tuberculosis</i> (Mtb) targets is a strategic priority for global TB control efforts. This research direction offers a robust, evidence-based solution to the urgent, growing challenge of anti-TB drug resistance. Thioredoxin reductase (TrxB2, Rv3913) is an essential enzyme for the in vitro growth and survival of Mtb. At present, no anti-TB lead compounds targeting TrxB2 have been advanced for clinical translation, primarily due to challenges such as off-target drug side effects and insufficient target specificity. We employed a virtual screening approach to screen compounds from small-molecule libraries. We conducted further experiments, including enzyme activity assays and studies using overexpression strains, to verify the therapeutic target and mechanism of action of the hit compound. Using virtual screening, we discovered compound OSSL_632214, which exhibited potent anti-Mtb activity (MIC 3.13–6.25&#xa0;μg/mL) in vitro and showed no cross-resistance with existing anti-TB drugs. In this study, we report for the first time the potent in vitro anti-Mtb activity of OSSL_632214, which possibly acts by targeting TrxB2 and inducing lipid peroxidation in Mtb. These findings provide a theoretical basis for developing novel anti-TB drugs targeting TrxB2.</p> Key points <p>• <i>Development of anti-TB drugs based on the novel target TrxB2</i></p> <p>• <i>OSSL_632214 exhibits anti-Mycobacterium tuberculosis activity</i></p> <p>• <i>OSSL_632214 exhibits potential for the development of TrxB2-targeted drug</i></p>