Different studies describe the anti-inflammatory effects of Scrophularia species, a medicinal plant widely used in folk medicine since ancient times. As knowledge regarding the anti-neoplastic properties of this species is rather limited, we investigated the influence of methanol extracts of different Scrophularia species on cell proliferation, cell death, and tumour cell intravasation through the lymph endothelial barrier. HL-60 leukaemia cells were treated with methanol extracts of different Scrophularia species leading to strong growth inhibition and high cell death rates. The expression of cell cycle regulators, oncogenes and cell death inducers was determined by Western blot analysis. Furthermore the effect of S. lucida was studied in an NF-κB reporter assay, and in a novel assay measuring 'circular chemo-repellent-induced defects' (CCID) in lymph endothelial monolayers that were induced by MCF-7 breast cancer spheroids. Methanol extracts of Scrophularia species exhibited strong anti-proliferative properties. S. floribunda extract inhibited G2/M- and later on S-phase and S. lucida inhibited S-phase and in both cases this was associated with the down-regulation of c-Myc expression. Extracts of S. floribunda and S. lucida led to necrosis and apoptosis, respectively. Furthermore, S. lucida, but not S. floribunda, effectively attenuated tumour cell intravasation through lymph endothelial cell monolayers, which correlated with the inhibition of NF-κB. S. lucida exhibited promising anti-neoplastic effects and this was most likely due to the down-regulation of various cell cycle regulators, proto-oncogenes and NF-κB and the activation of caspase-3.