The sequence of events during the pre-parasitic and parasitic stages of the life cycle of Globodera rostochiensis, starting from hatching until host invasion and infection, is of major importance. Many of the physiological changes are initiated in the pre-parasitic stage in response to the influences exerted by the root diffusates of the host plant. The objectives of this study were to investigate whether root diffusates and extracts of green manures from Brassicaceae, in particular yellow mustard, fodder radish and rapeseed, have a direct effect on the pre-parasitic stage of G. rostochiensis. Unhatched second-stage juveniles (J2) were exposed to root diffusates and extracts from roots or above-ground parts of the green manures to assess: i) changes in eggshell permeability; ii) changes in diameter of the dorsal gland nucleolus; and iii) the activation of transcription in the dorsal and subventral pharyngeal glands. The 24 h exposure of unhatched J2 to the green manures did not indicate an alteration in eggshell permeability. The diameter of the dorsal gland nucleolus increased significantly after exposure to diffusates but not to extracts of the green manures, although the size in that case still did not exceed the measurements in J2 exposed to tomato root diffusates. The expression of the gene IVg9 in the dorsal gland was not detected after any treatment. However, transcriptional activation in the subventral pharyngeal glands was observed. Although the maximum expression of the cellulase gene was found in J2 exposed to tomato root diffusates, the expression in J2 exposed to root extracts of green manures was higher than in J2 exposed to root diffusates and extracts of above-ground plant parts. The potential of the investigated brassicaceous green manures to influence the pre-parasitic stage of G. rostochiensis is discussed.