Methanolic extracts of Aloe barbadensis Miller inner leaf gel were fractionated by RP-HPLC and the resultant fractions were tested for inhibitory activity against a panel of bacteria and fungi. Five fractions were identified as having antimicrobial activity. Fraction 1 had the broadest antibacterial activity, being capable of inhibiting growth of both Gram-positive and Gram-negative bacteria as well as inhibiting growth of a nystatin resistant strain of the fungus Aspergillus niger. Fraction 1 had similar UV spectral properties as aloe emodin and was chromatographically identical to the pure compound. The other fractions tested were much more selective in their antimicrobial activities, being only capable of inhibiting the growth of specific Gram-negative rod bacteria. Two of these antimicrobial fractions were identified by ESI mass spectroscopy as being isomers of 8-C-߭D-[2-0-(E)-coumaroyl] glucopyranosyl -2-[2-hydroxy]-propyl-7-methoxy-5-methylchromone.