Bitetracenomycin A (<b>1</b>) and its diastereomers [(±)-bitetracenomycin B, (±)-<b>2</b>] were discovered from the cultures of <i>Streptomyces</i> sp. HDN154193. Compounds <b>1</b> and (±)-<b>2</b> were the first tetracenomycin dimers obtained from a natural source with sp<sup>3</sup> methine protons at the bridge positions (C-12/12'), which also exhibited broad-spectrum antibacterial activity. The racemate (±)-<b>2</b> was semisynthesized and separated into enantiomers (+)-<b>2</b> and (-)-<b>2</b>, and the absolute configurations were determined by specific rotation and ECD data. These metabolites exhibited potent antibacterial activity especially against drug-resistant strains (MRSA and MRCNS) with MIC values ranging from 1.0 to 1.9 μg/mL.