The essential oil composition, antimicrobial, anti-inflammatory and cytotoxic activities of 17 indigenous Agathosma species (18 samples) were investigated in order to validate their use in traditional healing. The results were related to the chemical composition of the essential oils as determined by GC and GC/MS. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) method on four pathogens: Staphylococcus aureus (ATCC 12600), Bacillus cereus (ATCC 11778), Klebsiella pneumoniae (NCTC 9633) and Candida albicans (ATCC 10231). The anti-inflammatory activity was evaluated using the 5-lipoxygenase assay while the cytotoxic activity was determined using the MTT (3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl-2H-tetrazolium bromide) cellular viability assay. The antimicrobial assay revealed that the most active oil against C. albicans was A. collina (MIC value of 3 mg/mL) whilst the most active oils against B. cereus were A. crenulata and A. pungens (MIC values of 3 mg/mL). Nine of the species had MIC values of 4 mg/mL against the Gram-positive pathogen S. aureus. The oils showed less activity against the Gram-negative pathogen K. pneumoniae. All the oils exhibited good in vitro anti-inflammatory activity with A. collina being the most potent (IC50 value of 25.98 ± 1.83 μg/mL). The results show that the oils were strong inhibitors of the enzyme 5-lipoxygenase. The oils proved to be toxic in the MTT assay displaying IC50 values of <0.0001 μg/mL, which were relatively toxic when compared to a plant-derived compound such as quinine (IC50 value of 0.08 ± 0.03 μg/mL). The results revealed some relationships between the major components, some bioactivities and toxicities. The oils were found to differ qualitatively and quantitatively in compositions and their analysis resulted in the identification of a total of 322 compounds in 18 of the samples.