The aerial parts of 16 individual Artemisia afra plants from four natural populations were hydrodistilled and the essential oil analyzed by GC/MS. The oil composition varied quantitatively and qualitatively within and between natural populations and showed no correlation to the geographical distribution. The antimicrobial activity was demonstrated by means of time-kill methodology using the respiratory pathogens C. neoformans and K pneumoniae. Antimicrobial activity was most prominent within 10 min at concentration 0.75% for K pneumoniae and within 60 min at concentration 1% for C. neoformans. Investigation of the four major compounds most abundant in the A. afra oil (artemisia ketone, 1,8-cineole, alpha- and beta-thujone) indicated minimal antimicrobial activity when investigated independently and in various combinations against K pneumoniae.