Volatile oils from the leaves, flowers and rhizomes of Dietes bicolor (Steud.) Sweet ex Klatt (Iridaceae) were analyzed using GLC/FID and GLC/MS. A total of 84 compounds were identified accounting for 94.65, 95.63 and 87.09% in the hydrodistilled oils from flowers, leaves and rhizomes, respectively. Spathulenol (48.44%) represented the major component in the leaf oil, followed by dihydro-edulan I (6.25%), cubenol (6.00%) and τ-cadinol (5.90%). For the flower and rhizome oils, fatty acids, their esters, aliphatic hydrocarbons and their derivatives predominate. The antimicrobial activity of both leaf and flower oils was investigated against four bacteria in addition to four fungi using the micro-broth dilution method. The leaf oil showed a more potent antimicrobial activity as compared to the flower oil against most of the assessed bacteria and fungi, with higher activities against Gram- positive organisms showing MIC values of 115 and 460 μg/ml for Bacillus subtilis and Streptococcus pneumonia, respectively. Gram-negative bacteria were generally less susceptible (MIC > 2 mg/ml for both oils against Escherichia coli) and being completely ineffective against Pseudomonas aeruginosa. A relevant antifungal potency of the leaf oil against Geotrichum candidum and Syncephalastrum racemosum was also observed with MIC values of 115 and 920 μg/ml, respectively.