The essential oil composition and antimicrobial activity of Osmitopsis asteriscoides, a medicinal plant used in traditional herbal preparations in South Africa has been investigated. Three different antimicrobial methods (disc diffusion, minimum inhibitory concentration by micro-titer plate and time-kill studies) were comparatively evaluated against Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. A preliminary screening was done using the disc diffusion method on nine bacterial and four fungal isolates. Minimum inhibitory concentrations showed some correlation with the disc diffusion method. However, time-kill studies appear to be a more superior method for determining antimicrobial activity of volatile compounds such as essential oils. Two moderately susceptible and one resistant organism were selected to further demonstrate the variability between the three methods. The antimicrobial activity of the essential oil, tested by means of time-kill methodology at concentrations ranging from 0.5 to 2% (v/v) indicate a strong fungicidal activity against Candida albicans and the oil was also found to be bacteriostatic against Staphylococcus aureus in a concentration-dependent manner. The essential oil rapidly reduced viable counts of Pseudomonas aeruginosa, but regrowth was noted after 240 min. The results have been generated in duplicate in separate microbiology laboratories using different time-kill methods and the results are congruent. The two major essential oil components camphor and 1,8-cineole were investigated indicating the positive antimicrobial efficacy of 1,8-cineole independently and in combination with camphor. In addition to (−)-camphor and 1,8-cineole, 40 compounds were identified by GC-MS in the hydro-distilled essential oil. The high concentration of cineole and camphor and their synergistic effect is presented as a possible explanation for the traditional use of Osmitopsis asteriscoides for treating microbe-related illnesses.