Lydiamycin A (1) is a previously reported piperazic acid-bearing cyclic peptide from Streptomyces. The absolute configuration of lydiamycin A has not been fully determined despite the fact that multiple total syntheses have been reported. The absolute configuration of 1 was reinvestigated by the advanced Marfey's method, chemical derivatization, and quantum-mechanics-based computational analysis, eventually resulting in a structural revision and establishment of the complete configuration. Lydiamycin A displayed weak antituberculosis activity in vitro.