The modern chemotherapy era started with Fleming's discovery of benzylpenicillin. He demonstrated that benzylpenicillin did not kill In this study, we found that >64 mg/liter of static benzylpenicillin concentrations killed 1.16 to 1.43 log CFU/ml below starting inoculum of extracellular and intracellular over 7 days. When we added the β-lactamase inhibitor avibactam, benzylpenicillin maximal kill ( ) of extracellular log-phase-growth was 6.80 ± 0.45 log CFU/ml at a 50% effective concentration (EC ) of 15.11 ± 2.31 mg/liter, while for intracellular it was 2.42 ± 0.14 log CFU/ml at an EC of 6.70 ± 0.56 mg/liter. The median penicillin (plus avibactam) MIC against South African clinical strains (80% either multidrug or extensively drug resistant) was 2 mg/liter. We mimicked human-like benzylpenicillin and avibactam concentration-time profiles in the hollow-fiber model of tuberculosis (HFS-TB). The percent time above the MIC was linked to effect, with an optimal exposure of ≥65%. At optimal exposure in the HFS-TB, the bactericidal activity in log-phase-growth was 1.44 log CFU/ml/day, while 3.28 log CFU/ml of intracellular was killed over 3 weeks. In an 8-week HFS-TB study of nonreplicating persistent , penicillin-avibactam alone and the drug combination of isoniazid, rifampin, and pyrazinamide both killed >7.0 log CFU/ml. Monte Carlo simulations of 10,000 preterm infants with disseminated disease identified an optimal dose of 10,000 U/kg (of body weight)/h, while for pregnant women or nonpregnant adults with pulmonary tuberculosis the optimal dose was 25,000 U/kg/h, by continuous intravenous infusion. Penicillin-avibactam should be examined for effect in pregnant women and infants with drug-resistant tuberculosis, to replace injectable ototoxic and teratogenic second-line drugs.