Ethnobotanical field studies in the Graaff-Reinet and Murraysburg regions (southeastern Karoo) have revealed a wealth of traditional knowledge on medicinal plants and their uses amongst elderly people of Khoi-San and Cape Dutch decent. The materia medica includes at least 86 species, most of which appear to be still in everyday use. The use of exotic plants (12 species) and similarities with the Xhosa healing culture show that the traditional system is dynamic and adaptive. Medicines to treat problems of the stomach, back, kidneys, bladder, as well as colds and other minor ailments have a high frequency. Mixtures of different plants are often used. An overview of the most important plants and their uses is presented, which shows several interesting records that have hitherto remained undocumented. These include new uses, new vernacular names and new medicinal plants (Abutilon sonneriatum, Aloe striata, Eberlanzia spinosa, Helichrysum pumilio, Osteospermum herbaceum, Pachypodium succulentum, Peliostomum cf. origanoides, Pentzia punctata, Rhigozum obovatum and Stapelia olivaea). New records of plants that are locally commonly used (e.g. H. pumilio and O. herbaceum) confirm that the medical ethnobotany of the Karoo is incompletely recorded. Keywords: Cape Dutch culture, Ethnobotanical survey, Khoi-San culture, Medicinal plants, Phytotherapy, Southeastern Karoo.