Pathogenic microscopic organisms in source waters pose a risk to public health if water treatment plants do not remove them. It was thought that sensitive PCR tests could be developed to inform decision-making about the most appropriate treatment processes, and to check the absence of pathogens from drinking water. This research focussed on four pathogen classes: cryptosporidium, microcystis, adenovirus and ammonia oxidising bacteria; and evaluated six DNA extraction kits. The Qiagen kit was most cost-effective for extracting DNA and Promega To-Taq polymerase was best for carrying out the PCR test on pathogens in real-world water samples. Other components of the PCR tests that were developed included test controls and DNA standards. A test for each class of pathogen was established and written as a ‘Standard Operating Protocol’ (SOP) which was then applied in different laboratories around Australia. Between-laboratory comparison of results showed the developed PCR tests to be highly reproducible and reliable. They can now be added to the existing suite of tools used to minimise risks to public health.