The anti-Trypanosoma brucei brucei activity in vitro and in vivo of a lactone-rich fraction of Vernonia blumeoides leaves (VBLF) and its potential in alleviating trypanosome-induced anemia and organ damage were investigated. Gas chromatography-mass spectrometry (GC-MS) analysis of VBLF revealed the presence of a number of lactone-containing compounds. In an in vitro study, VBLF showed concentration-dependent activity and was further used to treat T. brucei brucei-infected rats. The VBLF treatments, especially at 300 mg/kg body weight (BW), significantly (P < 0.05) kept the parasites reduced during the entire experimental period compared with the infected untreated group. At the end of the experiment, the trypanosome-induced anemia and hepatic damage were significantly (P < 0.05) alleviated in all the VBLF treatment groups, but renal damage was only prevented in the 200 and 300 mg/kg BW treatment groups. Furthermore, the trypanosome-induced increase in the relative weights of liver, spleen and kidney were significantly (P < 0.05) alleviated by the 300 mg/kg BW VBLF treatment. It was concluded that orally administered VBLF, especially at 300 mg/kg BW, possessed antitrypanosomal activity and could alleviate parasite-induced anemia and organ damage.