Free-living amoebae (FLA) are ubiquitous in natural and engineered water systems and are associated with both opportunistic and non-opportunistic infections. Acanthamoeba, Balamuthia, Naegleria, and Vermamoeba vermiformis are important FLA and responsible for keratitis and meningoencephalitis. Some FLA are natural hosts and vehicles of opportunistic plumbing pathogens including the bacterium Legionella pneumophila. L. pneumophila sg1 and sg2-14 are the primary causative agent of Pontiac fever, an acute flu-like illness, and Legionnaires’ disease, an atypical pneumonia that is potentially fatal in immunocompromised and elderly people. Amoebae protect Legionella from disinfection procedures with both able to persist in engineered water system. The aim of this study was to investigate the presence of FLA and their co-occurrence with Legionella. A total of 140 samples, including shower water, basin water and biofilm samples were collected from Australian domestic and hospital water systems. These samples were examined using both molecular and culture-based methods. Microbe-specific qPCR assays of the samples demonstrated that 41% were positive for Legionella, 33% for L. pneumophila, 55% for Vermamoeba and 11% for Acanthamoeba gene markers. Only 7% were positive for culturable Legionella, which were identified as L. pneumophila sg1, sg2-14 and non-pneumophila Legionella. Altogether, 41% of samples contained culturable amoebae, which were identified as Acanthamoeba, Allovahlkampfia, Stenamoeba and Vermamoeba. The pathogenicity potential of the isolates was determined by thermotolerance, osmotolerance and bacterial predation assays. Vermamoeba demonstrated high thermotolerance and osmotolerance suggesting they were highly pathogenic strains. The bacterial predation assay showed that both Acanthamoeba and Vermamoeba harbour strong broad spectrum bacteriogenic activity against Gram-negative and Gram-positive test organisms. These results demonstrate the prevalence of Legionella-associated FLA is high in Australian engineered water systems. In conclusion, a better understanding of L. pneumophila-FLA interactions is required for the proper management of legionellosis and amoebic infections.