Twenty South African medicinal plant species were selected by conducting a literature review based on the relevant information of their reported traditional medicinal uses and scientific reports against Alzheimer's disease, dementia, anxiety, mental illness, depression, acetylcholinesterase inhibition, headache, epilepsy, convulsion, hysteria, and sedative effects. The goal of this study was to investigate the biological activity of the traditionally used medicinal plant extracts against Alzheimer's disease by in vitro screening of the extracts to determine their potential to decrease levels of Aβ42 protein. Different plant parts (leaves, stem, bark, and stalks) of twenty selected plants were collected from the Manie van der Schijff Botanical Garden, University of Pretoria. Plant parts were dried, ground and then extracted using DCM:MeOH (1:1). We measured the levels of β-amyloid precursor protein proteolytic products in HeLa cells stably transfected with APP carrying the Swedish mutation using ELISA. Of 33 plant extract 10 (30.3%) were found active based on the potential to significantly reduce the production of Aβ42. Amongst them extracts of leaves of Xysmalobium undulatum (Apocynaceae), leaves of Cussonia paniculata (Araliaceae) and leaves of Schotia brachypetala (Fabaceae) potently decreased the production of Aβ42 by 77.3 ± 0.5%, 57.5 ± 1.3%, and 44.8 ± 0.1%, respectively. X. undulatum and S. brachypetala enhanced non-amyloidogenic processing of β-amyloid precursor protein, thereby decreasing Aβ42 level. We also showed that C. paniculata induced the decrease of Aβ42 level through inhibiting APP processing. In addition, we isolated two cardenolides, compound [A] and [B], from X. undulatum and found that they potently decreased the Aβ42 production. These data suggest that the extract of X. undulatum, C. paniculata, and S. brachypetala have potential to be developed for Alzheimer's disease treatment. These active extracts and compounds are considered for further studies which examine their efficacy towards the reduction of Aβ42 through inhibiting APP process.