Dalbergia melanoxylonGuill. & Perr (Fabaceae) is widely utilized in the traditional medicine of East Africa, showing effects against a variety of ailments including microbial infections. Phytochemical investigation of the root bark led to the isolation of six previously undescribed prenylated isoflavanones together with eight known secondary metabolites comprising isoflavanoids, neoflavones and an alkyl hydroxylcinnamate. Structures were elucidated based on HR-ESI-MS, 1- and 2-D NMR and ECD spectra. The crude extract and the isolated compounds ofD. melanoxylonwere tested for their antibacterial, antifungal, anthelmintic and cytotoxic properties, applying established model organisms non-pathogenic to humans. The crude extract exhibited significant antibacterial activity against Gram-positiveBacillus subtilis(97% inhibition at 50 μg/mL) and antifungal activity against the phytopathogensPhytophthora infestans,Botrytis cinereaandSeptoria tritici(96, 89 and 73% at 125 μg/mL, respectively). Among the pure compounds tested, kenusanone H and (3R)-tomentosanol B exhibited, in a panel of partially human pathogenic bacteria and fungi, promising antibacterial activity against Gram-positive bacteria including methicillin-resistantStaphylococcus aureus(MRSA) andMycobacteriumshowing MIC values between 0.8 and 6.2 μg/mL. The observed biological effects support the traditional use ofD. melanoxylonand warrant detailed investigations of its prenylated isoflavanones as antibacterial lead compounds.