2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glutamate from fructose-6-phosphate and glutamine and plays an important role in chitin synthesis in fungi. We have established a new assay for fungal ketol-isomerase activity that is amenable to high throughput screening to identify enzyme inhibitors. Aspergillus fumigatus crude lysate was incubated with substrates and after incubation, reactions were terminated. Glutamate dehydrogenase, nitro blue tetrazolium chloride, phenazine methosulfate and β-NAD were added and the amount of glutamate formed by ketol-isomerase activity was determined by measuring OD_585nm. A feedback inhibitor, UDP-N-acetylglucosamine, of fungal ketol-isomerase was successfully detected by this assay (IC₅₀-0.48 mM). In a pilot scale screening, an active extract from an extremophilic bacterium was found, and the extract showed antifungal activity against A. fumigatus, Candida albicans and C glabrata.