The in vitro antiplasmodial activities and HIV neutralizing activities of six crude extracts from the leaves and roots of Berberis holstii were evaluated. The antiplasmodial activities were assessed against a chloroquine sensitive 3D7 strain of the malaria parasite, Plasmodium falciparum, and the HIV neutralizing activity were assessed against HBX2 and ZM53 pseudovirions. Parasite Lactate Dehydrogenase (pLDH) assay was used to measure parasite viability and Luciferase Reporter Gene assay was used to measure HIV neutralizing activity. The crude alkaloid extract of the roots showed the most potent anti-parasitic activity (IC50 = 0.17 μg/mL) with a selectivity index (SI) of 701.2 while the DCM/MeOH extract also showed good activity (IC50 = 2.33 μg/mL). The aqueous and total alkaloid extracts of the roots displayed significant anti-parasitic activity (IC50 = 3.64 and 3.85 μg/mL respectively). The aqueous extract of the leaves displayed good HIV neutralizing activity (IC50 = 5.84 μg/mL) against the HXB2 pseudo virus. All the extracts tested displayed low cytotoxicity against TZM-bl cells.