We report the isolation and characterization of three new nybomycins (nybomycins B-D, <b>1</b>-<b>3</b>) and six known compounds (nybomycin, <b>4</b>; deoxynyboquinone, <b>5</b>; α-rubromycin, <b>6</b>; β-rubromycin, <b>7</b>; γ-rubromycin, <b>8</b>; and [2α(1<i>E</i>,3<i>E</i>),4β]-2-(1,3-pentadienyl)-4-piperidinol, <b>9</b>) from the Rock Creek (McCreary County, KY) underground coal mine acid reclamation site isolate <i>Streptomyces</i> sp. AD-3-6. Nybomycin D (<b>3</b>) and deoxynyboquinone (<b>5</b>) displayed moderate (<b>3</b>) to potent (<b>5</b>) cancer cell line cytotoxicity and displayed weak to moderate anti-Gram-(+) bacterial activity, whereas rubromycins <b>6</b>-<b>8</b> displayed little to no cancer cell line cytotoxicity but moderate to potent anti-Gram-(+) bacterial and antifungal activity. Assessment of the impact of <b>3</b> or <b>5</b> cancer cell line treatment on 4E-BP1 phosphorylation, a predictive marker of ROS-mediated control of cap-dependent translation, also revealed deoxynyboquinone (<b>5</b>)-mediated downstream inhibition of 4E-BP1p. Evaluation of <b>1</b>-<b>9</b> in a recently established axolotl embryo tail regeneration assay also highlighted the prototypical telomerase inhibitor γ-rubromycin (<b>8</b>) as a new inhibitor of tail regeneration. Cumulatively, this work highlights an alternative nybomycin production strain, a small set of new nybomycin metabolites, and previously unknown functions of rubromycins (antifungal activity and inhibition of tail regeneration) and also provides a basis for revision of the previously proposed nybomycin biosynthetic pathway.