Mentha longifolia (L.) L. subsp. polyadena Briq. was collected from eight localities in southern Africa for a study of the chemical composition and antimicrobial activity. The essential oils were obtained by hydrodistillation and analysed by GC and GC/MS and a cluster analysis was performed on the oil dataset. From eight samples (representing eight natural populations), two major chemotypes were identified: a menthofuran-rich type (51-62%); and a cis-piperitone
oxide (15-36%) and piperitenone oxide-rich type (15-66%). The constituent analysis showed quantitative variation with higher amounts of oxygen-containing monoterpenes ranging from 57% to 90% whilst the sesquiterpene hydrocarbons ranged from 4% to 17%. The oil from the different geographical areas mostly showed moderate antimicrobial activity against Staphylococcus aureus, S. epidermidis, Bacillus cereus, Moraxella catarrhalis, Yersinia enterocolitica and Enterococcus faecalis. The oils were generally inactive against Escherichia coli and Salmonella typhimurium. Candida albicans and Cryptococcus neoformans indicated highest sensitivities for oil samples from Komukwane and Prins Albert. These results may in part provide scientific evidence for the extensive use of Mentha longifolia in traditional healing.