Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish be- tween viable and dead cells. In this study a novel approach combining the DNA intercalat- ing dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal ac- tivity of the veterinary antiseptic F10! Antiseptic Solution. This disinfectant killed B. den- drobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.