A Global Natural Products Social (GNPS) molecular network guided investigation into the chemistry of the Australian plant pasture-derived Streptomyces sp. CMB-PB041 led to the discovery, structure elucidation and assignment of absolute configurations to a series of new members of Class II macrocyclic spirotetronate polyketides family, glenthmycins A–M (1–13). Intriguing base catalysed rapid intramolecular trans-esterification was detected within the regio-isomers 3/6/8, 4/7/9 as well as 5/19/20. The structure activity relationship analysis carried out on 1–13 and the semi-synthetic analogues 14 and 21–26 revealed a promising Gram +ve antibacterial pharmacophore, effective against methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE), but with no detectable cytotoxicity to eukaryotic cells (i.e. fungal and human carcinoma). Of particular note, the semi-synthetic analogue glenthmycin K 9-valerate (26) was unique among glenthmycins in potently inhibiting growth of the full panel of Gram +ve pathogens (IC50 0.2–1.6 uM).