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Summary: The root extract of Peucedanum ostruthium (PO-E) was identified as a promising antibacterial source from a screening of 158 extracts against Staphylococcus aureus. It has also recently been shown to significantly decrease the survival of the nematode Caenorhabditis elegans. We used the biochemometric approach ELINA to investigate the phytochemical characteristics of the multicomponent mixture PO-E to identify the anti-infective constituent(s) targeting S. aureus and C. elegans. 1H NMR spectra of PO-E-derived microfractions were correlated with their respective bioactivity data. Heterocovariance analyses unambiguously identified ostruthin as an anti-staphylococcal constituent, which potently also inhibited Enterococcus spp.. ELINA demonstrated that anthelmintic activity was due to a combinatorial effect of ostruthin and isoimperatorin. A C. elegans-based survival and motility assay confirmed that isoimperatorin, imperatorin, and verapamil modulated the susceptibility of ostruthin. The combinatorial effect of these natural products was shown in larvae studies to be related to the function of the nematodes’ efflux pump.