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Indigofera daleoides Benth. ex Harv. & Sond. (Fabaceae) is used by traditional healers in Limpopo Province for treatment of diarrhoea. The bioassay-guided fractionation of the ethylacetate extract from I. daleoides led to the isolation of two pure compounds which were identified as compound 1 (6,2-O-[3-nitropropanoyl-β-D-glucopyranose]) and compound 2 with a novel skeleton (6,3′,4′-trihydroxyflavan 5′-O-glucopyranoside). These compounds were isolated for the first time from I. daleoides. Isolated compounds were tested for antibacterial and cytotoxic activities in vitro using micro dilution method and sodium-2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, respectively. The results showed that compound 2 (flavonoid), isolated from I. daleoides, exhibited the highest antibacterial activity against S. aureus, S. typhy, V. cholera, E. coli and S. dysentery with MIC values ranging between 25–50 μg/ml. Compound 1 (nitrocompound) was found to be less active than compound 2 (flavonoid) against most of the bacteria tested. Staphylococcus aureus had higher susceptibility to compound 2 (flavonoid) as compared to the other bacteria tested. Compound 1 (nitrocompound) and 2 (flavonoid) isolated from I. daleoides had relatively high fifty per cent inhibitory concentration (IC50 values) (349.4 and 364.5 μg/ml, respectively) as compared to the crude extract (IC50 254 μg/ml), thus suggesting these compounds are less cytotoxic to Vero cells than the crude ethanol extract. The results of this study suggest possible antidiarrhoeal potential of these compounds, especially compound 2 (flavonoid) from I. daleoides which need to be further investigated. This is the first report on the antibacterial activity and cytotoxicity of purified compounds from I. daleoides.