Monitoring the changes that occur to water during distribution is vital to ensure water safety. In this study, the biological stability of reverse osmosis (RO) produced drinking water, characterized by low cell concentration and low assimilable organic carbon, in combination with chlorine disinfection was investigated. Water quality at several locations throughout the existing distribution network was monitored to investigate whether microbial water quality changes can be identified. Results revealed that the water leaving the plant had an average bacterial cell concentration of 10³ cells/mL. A 0.5–1.5 log increase in bacterial cell concentration was observed at locations in the network. The residual disinfectant was largely dissipated in the network from 0.5 mg/L at the treatment plant to less than 0.1 mg/L in the network locations. The simulative study involving miniature distribution networks, mimicking the dynamics of a distribution network, fed with the RO produced chlorinated and non-chlorinated drinking water revealed that distributing RO produced water without residual disinfection, especially at high water temperatures (25–30 °C), poses a higher chance for water quality change. Within six months of operation of the miniature network fed with unchlorinated RO produced water, the adenosine triphosphate (ATP) and total cell concentration (TCC) in the pipe biofilm were 4 × 10² pg ATP/cm² and 1 × 10⁷ cells/ cm². The low bacterial cell concentration and organic carbon concentration in the RO-produced water did not prevent biofilm development inside the network with and without residual chlorine. The bacterial community analysis using 16S ribosomal RNA (rRNA) gene sequencing revealed that mesophilic bacteria with higher temperature tolerance and bacteria associated with oligotrophic, nutrient-poor conditions dominated the biofilm, with no indication of the existence of opportunistic pathogenic species. However, chlorination selected against most bacterial groups and the bacterial community that remained was mainly the bacteria capable of surviving disinfection regimes. Biofilms that developed in the presence of chlorine contained species classified as opportunistic pathogens. These biofilms have an impact on shaping the water quality received at the consumer tap. The presence of these bacteria on its own is not a health risk indicator; viability assessment and qPCRs targeting genes specific to the opportunistic pathogens as well as quantitative microbiological risk assessment (QMRA) should be included to assess the risk. The results from this study highlight the importance of implementing multiple barriers to ensure water safety. Changes in water quality detected even when high-quality disinfected RO-produced water is distributed highlight microbiological challenges that chlorinated systems endure, especially at high water temperatures.