The increasing drug resistance of Mycobacterium tuberculosis (Mtb), coupled with the limited availability of effective anti-tuberculosis medications, poses significant challenges for the management and treatment of tuberculosis (TB). Globally, non-tuberculous mycobacteria (NTM) infections are increasing, with Mycobacterium avium complex and Mycobacterium abscessus (Mab) being the most common in labs and having few treatment options. There’s an urgent need for innovative therapies against Mtb and NTM that are effective and have minimal side effects. The study evaluated the in vitro efficacy of JIB-04, a Jumonji histone demethylase inhibitor, against Mtb, Mab, and multidrug-resistant (MDR) clinical isolates using the minimum inhibitory concentration (MIC) assay. We also determined the minimum bactericidal concentrations (MBCs) of JIB-04 against the H37Rv and H37Ra strains. A time-kill assay was performed to assess the comparative efficacy of JIB-04 and rifampicin against H37Ra. Additionally, the study investigated the impact of JIB-04 on biofilm formation and the persistence of H37Ra over extended periods. Our findings demonstrated a substantial inhibitory effect of JIB-04 on the growth of Mab, Mtb, and MDR clinical isolates. JIB-04 showed bactericidal effects at twice the MIC, outperforming rifampicin in reducing viable cell counts over 8 days. It showed moderate cytotoxicity to mammalian cells but effectively inhibited biofilm formation. In our anoxia model, JIB-04 induced a significant, concentration-dependent reduction in bacterial load. JIB-04 is a promising candidate for the treatment of MDR tuberculosis.