A combined approach based on quantitative and nested polymerase chain reaction (qPCR and nPCR, respectively) has been set up to detect and quantify the unculturable endobacterium Candidatus Glomeribacter gigasporarum inside the spores of its fungal host Gigaspora margarita. Four genes were targeted, two of bacterial origin (23S rRNA gene and rpoB) and two from the fungus (18S rRNA gene and EF1-alpha). The sensitivity of the qPCR protocol has proved to be comparable to that of nPCR, both for the fungal and the bacterial detection. It has been demonstrated that the last detected dilution in qPCR corresponded, in each case, to 10 copies of the target sequences, suggesting that the method is equally sensitive for the detection of both fungal and bacterial targets. As the two targeted bacterial genes are predicted to be in single copy, it can be concluded that the detection limit is of 10 bacterial genomes for each mixture. The protocol was then successfully applied to amplify fungal and bacterial DNA from auxiliary cells and extraradical and intraradical mycelium. For the first time qPCR has been applied to a complex biological system to detect and quantify fungal and bacterial components using single-copy genes, and to monitor the bacterial presence throughout the fungal life cycle.