From the anti-TB active fractions of the inner stem bark of Oplopanax horridus, two new heterocyclic nerolidol derivatives, 3,10-epoxy-3,7,11-trimethyldodeca-1,6-dien-11-ol, named neroplomacrol (1), and rel-(3S,6R,7S,10R)-7,10-epoxy-3,7,11-trimethyldodec-1-ene-3,6,11-triol, named neroplofurol (2), were isolated together with oplopandiol (3), falcarindiol (4), and sesamin (5). Extensive spectroscopic analysis revealed that 1 possesses a novel 3,10-oxanonacyclic ring system. Computer-iterated full spin system analysis led to the generation of (1)H NMR fingerprints that will facilitate future dereplication of analogues by providing characteristic spin-spin coupling patterns. The full spin analysis of 5 revealed asymmetric coupling patterns among the chemically equivalent spins, thus confirming the magnetic asymmetry of 5. It was further demonstrated that (1)H NMR fingerprints and MS data enable dereplication of isolates at a submilligram levels including their relative configuration. Countercurrent concentration of the anti-TB activity of the ethnobotanical O. horridus versus the Mycobacterium tuberculosis Erdman strain led to polyynes 3 and 4 as main anti-TB active principles. Their synergistic behavior is linked to a complex fraction containing the new nerolidiol sesquiterpenes, 1 and 2, as phytochemical marker compounds.