Thirty-five thousand actinomycete extracts were screened for anti-Mycobacterium tuberculosis (M. tb) activity, followed by C18 cartridge fractionation of 37 prioritized extracts. Based on MICs against replicating and nonreplicating M. tb, and IC50 values against Vero cells to generate selectivity indices, seven fractions from seven different strains were selected for further examination. When cultured in G.S.S. media and extracted with ethyl acetate, the Streptomyces hygroscopicus strain ECUM 14046 yielded an extract with promising anti-M. tb activity and a well-defined chromatographic profile. Fractionation by preparative HPLC and subsequent structure elucidation of two active fractions using 1D- and 2D-NMR and MS methods revealed the presence of two cyclohexapeptides, hytramycins V and I, each containing three unusual piperazic acid moieties. The use of (1)H iterative full spin analysis (HiFSA) on both hytramycins confirmed that quantum mechanics-simulated spectra match the experimental data, and all J(H,H) and δH values are consistent with the proposed structures. The absolute configuration of each amino acid moiety was determined by Marfey's method. The MICs against replicating and, more importantly, nonreplicating M. tb fall into the range of some existing second-line anti-TB drugs, such as streptomycin and capreomycin, respectively. The activities were maintained against M. tb strains that represent the major global clades, as well as H37Rv-isogenic strains that are resistant to individual clinical anti-TB drugs.