Frequently present in pancreatic, colorectal and non-small cell lung carcinomas, oncogenic mutant K-Ras must be localised to the plasma membrane (PM) to be functional. Inhibitors of K-Ras PM localisation are therefore putative cancer chemotherapeutics. By screening a microbial extract library in a high content cell-based assay we detected the rare oligomycin class of Streptomyces polyketides as inhibitors of K-Ras PM localisation. Cultivation and fractionation of three unique oligomycin producing Streptomyces strains yielded oligomycins A-E () and 21-hydroxy-oligomycin A (), together with the new 21-hydroxy-oligomycin C () and 40-hydroxy-oligomycin B (). Structures for were assigned by detailed spectroscopic analysis. Cancer cell viability screening confirmed were cytotoxic to human colorectal carcinoma cells (IC50 > 3 μM), and were inhibitors of the ABC transporter efflux pump P-glycoprotein (P-gp), with being comparable in potency to the positive control verapamil. Significantly, oligomycins proved to be exceptionally potent inhibitors of K-Ras PM localisation (Emax 0.67-0.75 with an IC50 ∼ 1.5-14 nM).