Landolphia myrtifolia is a plant utilized traditionally to treat fatigue in Madagascar. The objective of this study was to evaluate the antifatigue activity of Landolphia myrtifolia extract in mice subjected to both short- and long-duration physical exertion. A total of 20.2 g of defatted ethanolic extract was obtained from 500 g of Landolphia myrtifolia root powder by maceration in an ethanol-water mixture (80:20), yielding 4.04%. Phytochemical screening revealed a high content of flavonoids, tannins, and leucoanthocyanidins, with lower amounts of heterosides and polyphenols. In the hanging test, mice treated with the extract at doses of 100, 200, and 400 mg/kg showed significantly increased suspension times of 41.11 ± 0.42, 60.88 ± 0.51 and 87.55 ± 0.55 seconds, respectively, compared to 30 ± 1.41 seconds in the negative control group. The reference group exhibited a suspension time of 51.33 ± 0.55 seconds. In the forced swimming test, treated mice swam for 21.46 ± 0.40, 28.45 ± 0.84, and 54.62 ± 2.36 minutes at doses of 100, VBC200, and 400 mg/kg, respectively, significantly longer than the control group (8.62 ± 0.28 minutes, P < 0.05). The reference drug STIMOL induced a swimming time of 14.33 ± 0.37 minutes. Additionally, the distance traveled on the rotarod increased dose-dependently in treated mice (7.47 ± 0.19, 9.19 ± 0.27, and 17.47 ± 0.41 cm) compared to controls (2.69 ± 0.18 cm), while the reference group covered 4.358 ± 1.201 cm. These results indicate that Landolphia myrtifolia extract, supporting its potential as a natural antifatigue agent.