Fungal growth on solid foods can make them unfit for human consumption, but certain specialty foods require fungi to produce their characteristic properties. In either case, a reliable way of measuring biomass is needed to study how various factors (e.g. water activity) affect fungal growth rates on these substrates. Biochemical markers such as chitin, glucosamine or ergosterol have been used to estimate fungal growth, but they cannot distinguish between individual species in mixed culture. In this study, a real-time polymerase chain reaction (rt-PCR) protocol specific for a target fungal species was used to quantify its DNA while growing on solid food. The measured amount of DNA was then related to the biomass present using an experimentally determined DNA-to-biomass ratio. The highly sensitive rt-PCR biomass assay was found to have a wide range, able to quantify the target DNA within a six orders-of-magnitude difference. The method was used to monitor germination and growth of Penicillium chrysogenum spores on a model porous food (cooked wheat flour) at 25°C and different water activities of 0.973, 0.936, and 0.843. No growth was observed at 0.843, but lag, exponential and stationary phases were identified in the growth curves for the higher water activities. The calculated specific growth rates (μ) during the exponential phase were almost identical, at 0.075/h and 0.076/h for aw=0.973 and 0.936, respectively. The specificity of the method was demonstrated by measuring the biomass of P. chrysogenum while growing together with Aspergillus niger on solid media at aw=0.973.