A coffee beverage obtained from instant dark coffee that had been previously shown to possess high antibacterial activity, was acidified (pH 2) and extracted with ethyl acetate. After alkalinization (pH 12) the aqueous phase was re-extracted with the organic solvent. The acidic and basic extracts were evaporated to dryness and the aqueous phase freeze-dried. Residues were dissolved in sterile water and assayed for antibacterial activity against two reference bacteria (Staphylococcus aureus ATCC 25923 and Streptococcus mutans 9102). The acidic extract was found to be highly active and was separated by gel permeation chromatography (GPC) into five fractions. Fractions GPC4 and GPC5 were found to possess antibacterial activity: most of the activity was evident in fraction GPC5. These fractions were separated by RP-HPLC using a gradient elution with methanol water as mobile phase. Both GPC fractions gave an active subfraction with methanol-water (70:30, v/v). The experimental conditions used to separate the antibacterial compound that originates during the roasting process, indicate that it possesses low molecular mass (probably no more than 200 Da), weak acidic properties and an lambda(max) at 205 nm. The very small amount of this compound isolated from roasted coffee, indicates that it may be a very strong antibacterial agent.