A nested single-copy locus-based quantitative PCR (qPCR) assay and a multicopy locus-based qPCR assay were developed to estimate endophytic biomass of fungal root symbionts belonging to the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC). Both assays were suitable for estimation of endophytic biomass, but the nested assay was more sensitive and specific for PAC. For mycelia grown in liquid cultures, the correlation between dry weight and DNA amount was strong and statistically significant for all three examined strains, allowing accurate prediction of fungal biomass by qPCR. For mycelia colonizing cellophane or Norway spruce roots, correlation between biomass estimated by qPCR and microscopy was strain dependent and was affected by the abundance of microsclerotia. Fungal biomass estimated by qPCR and microscopy correlated well for one strain with poor microsclerotia formation but not for two strains with high microsclerotia formation. The accuracy of qPCR measurement is constrained by the variability of cell volumes, while the accuracy of microscopy can be hampered by overlapping fungal structures and lack of specificity for PAC. Nevertheless, qPCR is preferable because it is highly specific for PAC and less time-consuming than quantification by microscopy. There is currently no better method than qPCR-based quantification using calibration curves obtained from pure mycelia to predict PAC biomass in substrates. In this study, the DNA amount of A. applanata extracted from 15 mm of Norway spruce fine root segments (mean diameter, 610 microm) varied between 0.3 and 45.5 ng, which corresponds to a PAC biomass of 5.1 +/- 4.5 microg (estimate +/- 95% prediction interval) and 418 +/- 264 microg.