The linearmycin family of polyketides was originally classified as antifungal metabolites. However, in addition to antifungal activity, we previously found that linearmycins cause cellular lysis and colony degradation of the Gram-positive bacterium Bacillus subtilis. We recently showed that Streptomyces sp. strain Mg1 incorporates linearmycins into extracellular vesicles, which are capable of lysing B. subtilis. However, the mechanism of linearmycin-induced lysis was hitherto unexplored. Therefore, we sought to determine how linearmycin-laden vesicles cause lysis. In this study, we found that linearmycins inhibited the growth of all Gram-positive bacteria that we tested, but lysis was limited to some Bacillus species. Next, we found that linearmycin-induced lysis occurred even when cellular metabolism and growth were inhibited, which suggested that linearmycins possess the intrinsic capacity to lyse cells, unlike cell-wall targeting antibiotics. We showed that linearmycin exposure caused changes consistent with rapid depolarization of the B. subtilis cytoplasmic membrane, which was correlated with a loss of viability. Finally, using liposomes as in vitro membrane models, we demonstrated that linearmycins are capable of disrupting lipid bilayers without any other cellular components. Taken together, our results strongly indicate that the cytoplasmic membrane is the direct antibacterial target of linearmycins.