Botanicals represent a promising source of metabolites in the search for new biofungicides. In this context, this study aimed at evaluating the in vitro anti-oomycete activity of hop (Humulus lupulus L.) extracts and metabolites against Phytophthora infestans, an oomycete causing late blight disease in Solanaceae. Crude hydro-ethanolic extracts and dichloromethane sub-extracts of different parts (cones, leaves, stems and rhizomes) were characterized by UHPLC-UV–MS and some cone specialized metabolites were purified by CPC and preparative HPLC. A commercial hop cone essential oil was also analyzed by GC–MS. All extracts succeeded in inhibiting mycelial growth and spore germination with morphological alteration of the mycelium. Extracts of leaves showed a significant anti-oomycete activity compared to the extracts of cones, stems, and rhizomes. Moreover, no difference was noticed between the crude hydro-ethanolic extract and the dichloromethane sub-extract activity, except for leaves, with the apolar sub-extract being more active than the crude one. The extracts of cones succeeded in inhibiting more P. infestans than the essential oil, which appeared to be the less active evaluated modality. Some purified prenylated phenolic compounds also inhibited P. infestans although copper sulfate, a mineral fungicide control, was still more active. This study highlights the potential use of hop by-products as biofungicides to manage P. infestans.