Introduction Traditional healing is often the preferred form of therapy especially in rural and resource-limited communities. The extracts of plants are used to treat many diseases such as arthritis and chronic pain. Four medicinal plant species, namely, Acokanthera oppositifolia, Plantago lanceolata, Conyza canadensis and Artemisia vulgaris used in Southern Africa to treat pain and inflammation-related diseases were selected for evaluation in laboratory-based experiments. Methods The selected plant species were evaluated for phytochemical content, antioxidant and anti-inflammatory activities, as well as cytotoxicity effects against mammalian cells in culture. Results The results indicated that the n-hexane and chloroform extracts of P. lanceolata had the best antioxidant activities with an IC50=0.41 μg/mL. Also, the acetone extracts of P. lanceolata had 93.76% nitric oxide (NO) inhibition. However, the chloroform and n-hexane extracts of C. canadensis produced NO inhibition of 98.53% and 99.2%, respectively, at 100 μg/mL with IC50=17.69 μg/mL. Furthermore, the ethyl acetate extracts also had promising NO inhibitory activity (96.33%), but the cytotoxicity results with cell viabilities of 5.31%, 5.7% and 5.89%, respectively, suggested that the observed activity was due to a cytotoxic effect. Acetone extracts of C. canadensis were also cytotoxic at 30 µg/mL with 6.07-6.67% cell viabilities compared with the acetone extracts of P. lanceolata (99.57%). Conclusion The results partially validate the ethnomedicinal uses of the selected plant species used for inflammation-related conditions. However, because some of the extracts had potential cytotoxic effects, caution is advised in their use, especially those consumed orally.