Hospital water systems are a significant source ofLegionella, resulting in the potentially fatal Legionnaires’ disease. One of the biggest challenges forLegionellamanagement within these systems is that under unfavorable conditionsLegionellatransforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, onLegionellaspp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture andVermamoeba vermiformisandAcanthamoebaspecific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow®monitoring system. qPCR analysis demonstrated that 21.8% samples were positive forLegionellaspp., 21% forL. pneumophila, 40.9% forV. vermiformisand 4.2% forAcanthamoeba. All samples that wereLegionellaspp. positive using qPCR (22%) were also positive for VBNCLegionellaspp.;however, only 2.5% of samples were positive for culturableLegionellaspp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive forLegionellaspp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103CFU/L) were also significantly associated with high concentrations ofLegionellaspp. DNA, VBNCLegionellaspp./L. pneumophila(p< 0.01) andV. vermiformis(p< 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p< 0.01) increased the amount ofLegionellaspp. DNA, VBNCLegionellaspp./L. pneumophila, andV. vermiformis;however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion,Legionellaspp. (DNA and VBNC) was associated withV. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNCLegionellaspp. within a hospital water system. The high percentage of false negativeLegionellaspp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitorLegionellaspp. contamination within hospital water systems.