Callus and suspension cell cultures were successfully developed from Sutherlandia frutescens (Fabaceae), an endemic medicinal plant of South Africa. Two callus cell lines, originating from hypocotyl and cotyledon explants of in vitro seedlings under both dark and light conditions, showed intensive fresh weight accumulation with growth index ranging from 4.6 to 5.9. Suspension cell cultures induced from two callus lines had similar growth profiles and their growth index (15–18), specific growth rate (0.15–0.16 day−1), productivity (0.83–0.96 g/(l day)) and maximum biomass accumulation (16–18 g/l) remained relatively high for Fabaceae cell cultures during 27 sub-cultivations. Callus and suspension cell cultures showed similar profiles of secondary metabolites that were, however, different from leaves of greenhouse plants. Isoflavones were predominant in both callus and suspension cell cultures while flavonoids (sutherlandins) and triterpene glycosides of the cycloartane group (sutherlandiosides) were mostly found in leaves. Nineteen fatty acids (FA), both short- and very-long-chained (up to C25:0), were found in cell cultures. Linoleic and α-linolenic FA together comprised 60–64% out of total FA content in cell cultures followed by palmitic acid (18–25%). Extracts of suspension cell biomass exhibited antimicrobial activity against Staphylococcus aureus but were not effective against Pseudomonas aeruginosa. To the best of our knowledge, this is the first report on the induction, phytochemical composition and antimicrobial activity screening of S. frutescens suspension cell cultures which opens the door for their biotechnological application.