Two new withanolide glycosides, (20R,22R)-O-(3)-α-L-rhamnopyranosyl(1→4)-β-D- glucopyranosyl-1α,12βdiacetoxy-20-hydroxywitha-5,24-dienolide (3) and (20R,22R)-O-(3)-β-D-xylopyranosyl(1→3)-[β-D-xylopyranosyl (1→4)]-β-D-glucopyranosyl-1α-acetoxy-12β,20- dihydroxywitha-5,24-dienolide (4), were isolated from the leaves and root of Dunalia brachyacantha. Their aglycones, (20R,22R)-1α,12β-diacetoxy-3β,20dihydroxywitha-5,24-dienolide (or 1α,12β-diacetyldunawithagenine) and (20R,22R)-1α-acetoxy-3β,12β,20-trihydroxywitha-5,24-dienolide (or 1α-acetyl-12β-hydroxydunawithagenine), are novel. The known 18-acetoxywithanolide D (1) and 18-acetoxy-5,6-deoxy-5-withenolide D (2) were also isolated from the leaves. These last two compounds were shown to be responsible for the trypanocidal, leishmanicidal, and bactericidal activities manifested by the crude ethanolic extract. The structures were deduced from spectroscopic data and on the basis of chemical evidence.