In earlier reports from this laboratory, initial structural studies on the novel sulfur-containing antibiotic berninamycin A—a potent inhibitor of bacterial protein synthesis—were described, with structural subunits accounting for its total composition assigned via degradation products from acidic hydrolysis, methanolysis, and acetolysis. In the present communication, the total structure of berninamycin A (assigned as 1) was determined based on new compounds obtained by trifluoroacetolysis of the intact antibiotic and its sodium borohydride-reduced and catalytically hydrogenated derivatives. Trifluoroacetolysis of berninamycin A afforded three major compounds, including 3 (containing Hyval, Ox-A, and pyruvyl residues) and 5 (containing Thr, Ox-B, Deala, and Berninamycyl residues), which allowed assignment of subunits c and d. Reduction of berninamycin A with sodium borohydride followed by trifluoroacetolysis yielded compound 7, extending subunit c to e. Catalytic hydrogenation of berninamycin A produced dihydroberninamycin A and hexahydroberninamycin A; trifluoroacetolysis of hexahydroberninamycin A gave compound 8, leading to assignment of subunit f. Combining all subunits (including the Hyval-Ox-A sequence from prior work) and accounting for overlapping terminal Deala units of e and f, the total structure of berninamycin A was established as 1.