Erythrina abyssinica Lam. is an important medicinal plant growing in Sudan; its seeds were investigated for the first time for their alkaloidal constituents and biological activity. The in vitro cytotoxicity of the crude alkaloidal fraction (CAF) against the cell lines HeLa, Hep-G2, HEP-2, HCT116, MCF-7 and HFB4 showed promising activity, with IC₅₀ values of 13.8, 10.1, 8.16, 13.9, 11.4 and 12.2 µg mL⁻¹, respectively. Doxorubicin (positive control) showed in vitro cytotoxic activity with IC₅₀ values 3.64, 4.57, 4.89, 3.74, 2.97 and 3.96 µg mL⁻¹, respectively. Bioassay-guided fractionation and isolation of the CAF led to the isolation of five Erythrina alkaloids, identified as erythraline, erysodine, erysotrine, 8-oxoerythraline and 11-methoxyerysodine. These were evaluated for their in vitro cytotoxic activity against Hep-G2 which resulted in IC₅₀ values 17.60, 11.80, 15.80, 3.89 and 11.40 µg mL⁻¹, respectively. Furthermore, in vitro cytotoxic activity against HEP-2 was evaluated, which resulted in IC₅₀ values 15.90, 19.90, 21.60, 18.50 and 11.50 µg mL⁻¹, respectively. The CAF caused a reduction in the viability of mock-infected MT-4 cells with a CC₅₀ of 53 µM and a 50% protection of MT-4 cells against HIV-1 induced cytopathogeneticy with a EC₅₀ of >53 µM, compared with EFV as a positive control, which had a CC₅₀ of 45 µM and an EC₅₀ of 0.003 µM. We concluded that the isolated alkaloids were responsible for the anti-carcinogenic [corrected] actions of the plant extract previously reported in the literature.