This study was undertaken to compare the in vitro and ex vivo antibacterial activity of an ethanolic Harungana madagascariensis leaf extract (HLE) incorporated into poly (D,L-lactide-co-glycolide) nanoparticles (HLE-PLG-NP). Two concentrations of HLE (500 and 1,000 mg/mL) for the in vitro study and one concentration (500 mg/mL) for the ex vivo study were compared using two gram-positive bacterial strains (Micrococcus luteus and Staphylococcus epidermidis), and one gram-negative bacterial strain (Moraxella sp.). The ex vivo antibacterial activity was evaluated on S. epidermidis CIP 55109 (SE) using an artificial contamination method. SE was inoculated for 12 h onto human skin fragment surfaces treated for 5 min either with HLE loaded, unloaded PLG-NP, or HLE solution. In vitro, the two preparations inhibited completely the growth of all bacterial strains at 1,000 mg/mL. However, the HLE-PLG-NP had a significant antibacterial activity against SE (18.471.8-0.470.2 CFU/mL, Po0.05), and a marked antibacterial effect against M. luteus (ML) and Moraxella sp. (Msp) compared to HLE solution at 500 mg/mL. Ex vivo, HLE-PLG-NP at 500 mg/mL reduced viable bacteria (6.3-4.8 log 10 ), compared to the HLE solution (6.3-5.5 log 10 ) after 4 h artificial contamination (Po0.05). A thin layer chromatography study of both HLE solution and HLE-PLG-NP showed that among the seven components found on the chromatogram of the HLE solution, only two were present on the nanoparticles, one including a flavonoid heteroside fraction responsible for the antibacterial properties. The incorporation of the HLE into a colloidal carrier improved antibacterial performance. Drug Dev. Res. 65:26-33, 2005.