A simple bioautographic agar overlay assay using Candida albicans as the indicator organism for the detection and activity-guided fractionation of antifungal compounds by thin layer chromatography has been developed. Inhibition of fungal growth was assessed by the detection of dehydrogenase activity with thiazolyl blue (methylthiazolyltetrazolium chloride; MTT). A series of clinically used antimycotic agents were tested in order to determine the sensitivity of the assay. The compatibility of the agar overlay technique with chemically modified silica gel (Diol and RP-18) plates and with various organic solvents was evaluated. The methodology is also applicable to the search for antibacterial compounds, as shown with Bacillus subtilis as a test organism.