Pseudoperonospora cubensis causes some of the most devastating diseases of cucurbitaceous crops, while P. humuli is an important pathogen of hop (Cannabaceae). Although parasitic to different Angiosperm orders, these pathogens are highly similar, both in morphology and based on molecular comparisons. Considering the close relationship of P. humuli and P. cubensis, it was hypothesized that cross infectivity of the pathogens between their optimum hosts might be possible. Two strains of P. humuli and one of P. cubensis used in this study were able to reproduce on the two wild cucurbit relatives Bryonia dioica and Sicyos angulatus. Interestingly, limited infectivity of the P. cubensis strain to hop was also observed, and the P. humuli strain was also able to infest Cucumis sativus. The cross infections were verified by comparing the nuclear ribosomal-internal transcribed spacer sequences of newly produced sporangiophores from the different hosts. Morphologically the two pathogens could be distinguished by statistical analyses on their original hosts but these differences diminished on some alternate hosts. The ability of P. cubensis to infest the perennial Bryonia dioica and Humulus lupulus might offer a limited possibility for overwintering in temperate regions and needs to be evaluated in future studies. The ability of P. humuli to complete its life cycle on all the tested cucurbits provides evidence that the host jump across orders may continue and points to the high virulence potential of this pathogen.