Heteropyxis natalensis (Heteropyxidaceae) is traditionally used to treat respiratory disorders, and as a decongestant and antimicrobial agent. The seasonal variation of the hydrodistilled essential oil was investigated. Three trees in the Johannesburg Botanical Garden (Gauteng) indicated similar chemical profiles with fluctuation in the levels of the two major constituents (1,8-cineole and limonene). Little variation between the antimicrobial activity of seasonally collected samples was documented, with standard deviations of ± 0.3 to ± 3.3 depending on the pathogen studied. Moderate antimicrobial activity (3.0-16.0 mg/ml) was noted for most pathogens tested with Cryptococcus neoformans exhibiting the highest sensitivity (2.0-3.0 mg/ml). The chemogeographical variation of the oil composition from five of the seven distinct localities studied all contains 1,8-cineole and limonene as major constituents. The antimicrobial study of these samples indicated little variability between localities (standard deviation of ± 0.5 to ± 3.8). As observed in the seasonal variation study, C. neoformans displayed the highest sensitivity (0.5-2.0 mg/ml). One oil sample (Lagalametse), was distinctly different both chemically and microbiologically.