We describe a rapid and simple enumeration method for sulphate-reducing bacteria (SRB) from aquatic environments using a quantitative real-time PCR (qPCR) in combination with a rapid DNA isolation method. Enumeration of SRB in the sediment and water samples was performed by quantifying the copy number of the dsrA gene coding for the α-subunit of the dissimilatory sulphite reductase using real-time PCR with the SYBR Green I assay. Using dsrA-specific primers, we demonstrated that quantification of SRB in known numbers of SRB assemblages can be achieved. We compared DNA isolation methods using commercial DNA extraction kits and a published technique. We found two commercial kits were of advantage for extraction of DNA from water or sediment samples, where a large number of samples could be processed at the same time. We showed this newly developed qPCR technique targeting dsrA is rapid, simple and reproducible for the quantification of SRB numbers in situ and is superior to the use of culture-dependent techniques.