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Guiera senegalensis (Gs) is a well-known traditional medicinal plant in Africa, whose leaves extract and roots powder are used for treatment of diseases and wounds in Western Kordufan, Sudan. The aim of this study was to investigate the phytochemical analysis, toxicity, and the antifungal activity of Gs leaves extract. Extract of the leaves of Gs was tested for its antimicrobial activity against Stemphylium solani, Aspergillus flavus, Trichoderma viride, Penicillium Spp., Fusarium verticillatum, Cladosporium cladosporioides, and Fusarium solani. This present study showed that Gs leaves extract has no inhibition activity against all of the tested fungal strains. On the other hand, the toxicity test, which was conducted by using brine shrimp, suggests that Gs leaves extract is apparently not toxic. The phytochemical screening revealed that Gs leave extract contains alkaloids, flavonoids, terpenoids, tannin, carbohydrates, proteins, steroids, and saponins. The results of this preliminary investigation suggests that the medicinal plant extract may be safe to use as a drink for treatment of various diseases as has been practiced for years in the villages of Western Sudan. More research is needed to investigate if there is any side effect when the extract is taken orally. Further, the medicinal properties of the phytochemical compounds of Gs need to be further investigated.