Bacterial infections remain a significant threat to human health. Due to the emergence of widespread antibiotic resistance, development of novel antibiotics is required in order to ensure that effective treatment remains available. The aim of this study was to isolate and identify the fraction responsible for the antimicrobial activity in Tabernaemontana elegans (Stapf.) root extracts. The active fraction was characterised by thin layer chromatography (TLC) and gas chromatography – mass spectrometry (GC-MS). Antibacterial activity was determined using the broth micro-dilution assay and antimycobacterial activity using the BACTEC radiometric assay. Cytotoxicity of the crude extract and fractions was assessed against primary cell cultures; lymphocytes and fibroblasts; as well as a hepatocarcinoma (HepG2) and macrophage (THP-1) cell line using the Neutral Red uptake and MTT assays. The crude root extracts were found to contain a high concentration of alkaloids (1.2% w/w). GC-MS analysis identified the indole alkaloids, voacangine and dregamine, as major components. Antibacterial activity was limited to the Gram-positive bacteria and Mycobacterium species, with MIC values in the range of 64 – 256 ìg/ml. When combined with antibiotics, additive antibacterial effects were observed. Marked cytotoxicity to all cell lines tested was evident in the MTT and Neutral Red uptake assays, with IC50 values ranging between 1.11 – 9.81 ìg/ml. This study confirms the antibacterial activity of T. elegans and supports its potential for being investigated further for the development of a novel antibacterial compound.