In this study, we described the structure-activity relationships of substituted 3,5-dinitrophenyl tetrazoles as potent antitubercular agents. These simple and readily accessible compounds possessed high in vitro antimycobacterial activities against Mycobacterium tuberculosis, including clinically isolated multidrug (MDR) and extensively drug-resistant (XDR) strains, with submicromolar minimum inhibitory concentrations (MICs). The most promising compounds showed low in vitro cytotoxicity and negligible antibacterial and antifungal activities, highlighting their highly selective antimycobacterial effects. 2-Substituted 5-(3,5-dinitrophenyl)-2H-tetrazole regioisomers, which are the dominant products of 5-(3,5-dinitrophenyl)-1H-tetrazole alkylation, showed better properties with respect to antimycobacterial activity and cytotoxicity than their 1-substituted counterparts. The 2-substituent of 5-(3,5-dinitrophenyl)-2H-tetrazole can be easily modified and can thus be used for the structure optimization of these promising antitubercular agents. The introduction of a tetrazole-5-thioalkyl moiety at position 2 of the tetrazole further increased the antimycobacterial activity. These compounds showed outstanding in vitro activity against M. tuberculosis (MIC values as low as 0.03 μM) and high activity against non-tuberculous mycobacterial strains.