Chemical investigation of Australian pasture plant-derived <i>Streptomyces</i> sp. CMB-PB041, supported by miniaturized cultivation profiling and molecular network analysis, led to the isolation and characterization of 13 new macrocyclic spirotetronates, glenthmycins A-M (<b>1</b>-<b>13</b>), with structures assigned by detailed spectroscopic analysis, chemical degradation and derivatization, and mechanistic and biosynthetic considerations. Hydrolysis of glenthmycin B (<b>2</b>) yielded the aglycone <b>14</b>, whose structure and absolute configuration were secured by X-ray analysis, along with the unexpected amino sugar residues glenthose lactams A (<b>15</b>) and B (<b>16</b>), with Mosher analysis of <b>15</b> facilitating assignment of absolute configurations of the amino sugar. While the glenthmycins proved to be acid stable, treatment of isomeric glenthmycins (i.e., <b>3</b>, <b>6</b>, and <b>8</b>) with base catalyzed rapid intramolecular <i>trans</i>-esterification to regio-isomeric mixtures (i.e., <b>3</b> + <b>6</b> + <b>8</b>). Exposure of <b>5</b> to base achieved the same intramolecular <i>trans</i>-esterification and was instrumental in detecting and tentatively identifying two additional minor co-metabolites, glenthmycins N (<b>19</b>) and O (<b>20</b>). A structure-activity relationship analysis carried out on <b>1</b>-<b>13</b> and the semisynthetic analogues <b>14</b> and <b>21</b>-<b>26</b> revealed a promising Gram +ve antibacterial pharmacophore, effective against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) and vancomycin-resistant Enterococci (VRE), but with no detectable cytotoxicity to eukaryotic cells (i.e., fungal and human carcinoma). Of particular note, the semisynthetic analogue glenthmycin K 9-valerate (<b>26</b>) was unique among glenthmycins in potently inhibiting growth of the full panel of Gram +ve pathogens (IC<sub>50</sub> 0.2-1.6 μM). We conclude with an observation that any future evaluation of the antibacterial potential of glenthmycins and related macrocyclic spirotetronates may do well to include important soil-derived Gram +ve pathogens, such as <i>Bacillus anthrax</i>, <i>Clostridium botulinum</i>, and <i>Rhodococcus equi</i>, the causative agents of anthrax, botulism, and livestock pneumonia.