High-temperature environments and variations in hot water premise plumbing create unique microbial community characteristics that impact water safety. This study utilized a simulation platform for non-circulating hot water premise plumbing to investigate microbial growth under the effects of four specific hydraulic regimes: high variable flow (HIG), low variable flow (LOW), steady state (SS), and retention (R) conditions. After 91 days of operation, biofilm and water samples were collected from the simulation platform. The bacterial counts in the samples were measuerd, and the microbial community structure and the presence of potential pathogens genus were evaluated using 16S rRNA gene sequencing. The results showed that the R condition led to the highest microbial counts in both water and biofilm samples, while the continuously varying flow conditions, LOW and HIG, showed lower counts. Live bacteria demonstrated a propensity to colonize biofilms, exhibiting increased numbers and relative abundances of potentially pathogenic genera in the membrane samples. In addition, biofilms under varying flow conditions exhibited higher relative abundances of three typical potentially pathogenic genera, Pseudomonas spp., Legionella spp., and Mycobacterium spp., indicating a greater potential pathogenic risk.