This study represents a systematic chemical and biological study of the rufomycin (RUF) class of cyclic heptapeptides, which our anti-TB drug discovery efforts have identified as potentially promising anti-TB agents that newly target the caseinolytic protein C1, ClpC1. Eight new RUF analogues, rufomycins NBZ1-NBZ8 (<b>1</b>-<b>8</b>), as well as five known peptides (<b>9</b>-<b>13</b>) were isolated and characterized from the <i>Streptomyces atratus</i> strain MJM3502. Advanced Marfey's and X-ray crystallographic analysis led to the assignment of the absolute configuration of the RUFs. Several isolates exhibited potent activity against both pathogens <i>M. tuberculosis</i> H37Rv and <i>M. abscessus</i>, paired with favorable selectivity (selectivity index >60), which collectively underscores the promise of the rufomycins as potential anti-TB drug leads.