Two biotypes of the invasive species, Chromolaena odorata are known to be present in Africa, viz. the Asian/West African biotype (AWAB) and the southern African biotype (SAB). Although the phytochemistry, ethnomedicinal and ethnopharmacological relevance of the AWAB has been elucidated, the SAB plants have received little or no attention. This study investigated and compared the phytochemistry and pharmacological activities of two biotypes of C. odorata (AWAB and SAB). Antimicrobial activities of leaf extracts of the two biotypes and three different growth stages of the SAB were evaluated against several bacterial and fungal strains using a serial microdilution assay. Phytochemicals were determined through standard methods of analysis. Toxicity of the extracts of the different growth stages of the SAB was determined using the colorimetric MTT assay, while the mutagenicity assay was performed using the Ames test. The AWAB had the overall best antibacterial activity, while the SAB showed better antifungal activity. Results showed that young and mature non-flowering extracts of the SAB were the most active. AWAB contained the highest amount of phenolics and flavonoids while SAB contained the highest amount of tannins. Extracts of young SAB plants showed a low level of cytotoxicity and none of the extracts of the three growth stages were mutagenic. This is the first report suggesting that the SAB of C. odorata can be exploited as a source of medicine similar to the AWAB, in combating antimicrobial infections and other health problems.