The long-term treatment of tuberculosis (TB) sometimes leads to nonadherence to treatment, resulting in multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. Inadequate bioavailability of the drug is the main factor for therapeutic failure, which leads to the development of drug-resistant cases. Therefore, there is an urgent need to design and develop novel antimycobacterial agents minimizing the period of treatment and reducing the propagation of resistance at the same time. Here, we report the development of original and noncytotoxic polycationic phosphorus dendrimers essentially of generations 0 and 1, but also of generations 2–4, with pyrrolidinium, piperidinium, and related cyclic amino groups on the surface, as new antitubercular agents active per se, meaning with intrinsic activity. The strategy is based on the phenotypic screening of a newly designed phosphorus dendrimer library (generations 0–4) against three bacterial strains: attenuated Mycobacterium tuberculosis H37Ra, virulent M. tuberculosis H37Rv, and Mangora bovis BCG. The most potent polycationic phosphorus dendrimers 1G0,HCl and 2G0,HCl are active against all three strains with minimum inhibitory concentrations (MICs) between 3.12 and 25.0 μg/mL. Both are irregularly shaped nanoparticles with highly mobile branches presenting a radius of gyration of 7 Å, a diameter of maximal 25 Å, and a solvent-accessible surface area of dominantly positive potential energy with very localized negative patches arising from the central N3P3 core, which steadily interacts with water molecules. The most interesting is 2G0,HCl, showing relevant efficacy against single-drug-resistant (SDR) M. tuberculosis H37Rv, resistant to rifampicin, isoniaid, ethambutol, or streptomycin. Importantly, 2G0,HCl displayed significant in vivo efficacy based on bacterial counts in lungs of infected Balb/C mice at a dose of 50 mg/kg oral administration once a day for 2 weeks and superior efficacy in comparison to ethambutol and rifampicin. This series of polycationic phosphorus dendrimers represents first-in-class drugs to treat TB infection, could fulfill the clinical candidate pipe of this high burden of infectious disease, and play a part in addressing the continuous demand for new drugs.