The chemical profiles of essential oils isolated from nine populations of Helichrysum splendidum were obtained by gas chromatography. Plants were harvested in both summer and winter seasons in two provinces of South Africa. Essential oils originating from Limpopo Province were characterized by high levels of β-phellandrene, 1,8-cineole, δ-cadinene, α-cadinol, τ-cadinol, and β-pinene. These could be clearly distinguished through multivariate analysis from those collected in Mpumalanga Province. The Mpumalanga oils contained germacrene D, spathulenol and bicyclogermacrene as major constituents. Seasonal variations in the volatile compositions and yields were observed. A prediction model obtained through orthogonal projection to latent structures discriminant analysis was used to compare the essential oil profiles to that of a commercial H. splendidum sample, in addition to those reported for Zimbabwean specimens. One of the specimens from Zimbabwe differed substantially from all the others, indicating the existence of more chemotypes. The structures of the oil-bearing trichomes, as observed on the leaf surfaces using scanning electron microscopy, were investigated and are described for the first time. Since this investigation forms part of an ongoing screening of plants to identify natural alternatives to synthetic fungicides, the essential oils were tested in vitro against several economically important pathogens of fruit. Essential oils from Mpumalanga specimens totally inhibited the growth of Alternaria alternata from avocado and Colletotrichum gloeosporioides isolated from mango at a concentration of 500 μL/L in a toxic medium assay. The presence of high concentrations of germacrene D, spathulenol and bicyclogermacrene probably contributed to the antifungal properties of these essential oils.