Discolouration is the greatest cause of customer dissatisfaction with drinking water quality, potentially masking other failures, including microbial issues, which can impact public health and well-being. The theorised association between biofilms (complex microbial communities) and discolouration within drinking water distribution systems (DWDS) was explored, whilst studying the impact and interactions of seasonal temperature variations and hydraulic regime. Transferability of findings to operational DWDS was ensured by using a temperature controlled, full-scale distribution experimental facility. This allowed isolation of the factors of interest, with integration of physical, chemical and microbial analyses. Greater discolouration and biofilm cell accumulation was observed under warmer (summer, 16°C) temperatures compared to cooler (winter, 8°C), evidence of microbiology being an important driver in DWDS discolouration behaviour. Temperature was generally more influential upon discolouration and biofilm cell volumes than the shear stress imposed by the hydraulic regimes, which included three steady state and two varied flow patterns. However, the trends were complex, indicating interactions between the two parameters in governing microbial accumulation and discolouration. These results are important in informing sustainable management of our ageing DWDS infrastructure to deliver safe high quality drinking water. By providing new evidence that discolouration is a biofilm/microbiologically-mediated process, we can better understand the importance of targeting interventions to hotter seasons, and manipulating hydraulic conditions (which we can control), to minimise the long-term impacts of impending changing climates on water quality.