Background: Khaya senegalensis (Desr.) A. Juss. is a common component of the pharmacopeia’s of multiple African groupings which inhabit the areas in which it grows. Amongst these groups there is a myriad of medicinal uses in the treatment of a wide variety of bacterial, fungal and protozoal infections, as well as in the treatment of cancers. This study was undertaken to test K. senegalensis bark extracts for the ability to inhibit microbial and cancer cell growth, and thus to validate traditional African medicinal usage of this plant in treating a variety of diseases. Materials and Methods: K. senegalensis bark powder was extracted by both solvent maceration and subcritical fluid extraction (SFE). The extracts were tested for the ability to inhibit bacterial and G. duodenalis growth. Inhibition of Caco-2 and HeLa cancer cells was evaluated using MTS-based colorimetric cell proliferation assays. Toxicity was evaluated using an Artemia franciscana nauplii bioassay and GC-MS headspace analysis was used to identify phytochemical components. Results: K. senegalensis bark extracts displayed strong inhibitory activity against bacterial triggers of several autoimmune inflammatory diseases. The growth inhibitory activity of the methanolic and subcritical extracts was particularly noteworthy against P. mirabilis (MIC values of 185 and 211μg/mL, respectively against the reference strains). These extracts were similarly potent growth inhibitors of K. pneumoniae and A. baylyi and were moderate inhibitors (MIC >1000μg/mL) of P. aeruginosa and S. pyogenes growth. The methanolic and subcritical K. senegalensis extracts were also potent inhibitors of G. duodenalis (187 and 328μg/mL, respectively), as well as Caco-2 (268 and 470μg/mL, respectively) and HeLa carcinomas (155 and 174μg/mL, respectively). GC-MS analysis of the SFE extract revealed relative abundances of a variety of mono- and sesquiterpenoids. Furthermore, all K. senegalensis bark extracts were non-toxic in the Artemia franciscana toxicity assay, indicating their safety for therapeutic use. Conclusion: These studies validate traditional African therapeutic usage of K. senegalensis in the treatment of microbial infections, autoimmune inflammatory diseases and some cancers.