Many Gram -negative bacteria other than Pseudomonas aeruginosa have been implicated in waterborne outbreaks but standardised laboratory detection methods for these organisms have not been established. We aimed to establish laboratory testing methodologies for six waterborne pathogens; Acinetobacter spp, Burkholderia spp, Cupriavidus spp, Delftia acidovorans, Elizabethkingia spp and Stenotrophomonas maltophilia. Water samples were spiked by UK Health Security Agency labs and sent to the Glasgow Royal Infirmary lab for analysis. Water samples were spiked with either a pure culture of target organism or the target organism in water containing normal background flora, to ensure the methodology could identify organisms from a mixed culture. Volumes of 100 mL were filtered under negative pressure onto culture media and incubated at 30°C and 37°C. Incubation time was seven days with plates read on day two, day five and day seven. Further identification of colonies was undertaken using MALDI-TOF. Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar (TSA), Chocolate Bacitracin agar (BAC) and Pseudomonas selective agar (PSE). 30°C was the optimal temperature for isolation. Optimal incubation time was five days and MALDI-TOF identified all species tested reliably. The methodology described can reliably detect the six waterborne pathogens tested and can be utilised by labs involved in testing water samples during outbreak investigations.