This work was designed to study the transition from water treated with O3 and granular activated carbon to nanofiltered water with special regard to biofilm sloughing and turbidity, and to measure the extent of biofouling in an “old” network fed with non-chlorinated nanofiltered water. The experimental drinking water distribution system contained six loops in series, with a total hydraulic residence time (HRT) of 72 h. It was first fed with biologically treated water (GAC water) having organic carbon (1.6 mg-C litre−1, of which 22% was biodegradable) and microorganisms (1.12 × 105 cells ml−1, of which 7% was culturable after 15 days of incubation). It was then fed with nanofiltered water, which was contaminated with bacteria in transit from the nanofiltration unit to the network. This nanofiltered water 1 contained 0.23 mg-C litre−1 (biodegradable fraction < 0.1 mg-C litre−1) and 3.0 × 104 cells ml−1 (22% was culturable after 15 days). Finally, the system was fed with nanofiltered water 2 that contained few microorganisms (50 cells ml−1) and few nutrients (0.21 mg-C litre−1, biodegradable fraction < 0.1 mg-C litre−1). The changes in the water and biofilm quality in the experimental network fed successively with the three waters, and the water quality during transport through the network, were determined. The bacterial population in the system showed a gradual drop in the total number of cells and their culturability with the water changes. The total number of cells and the colony forming units (CFU) after 3–15 days decreased respectively about 10-fold and 100-fold after 1 year of feeding with nanofiltered water 2 compared to GAC water to reach 1.3 × 104 cells ml−1, 2 CFU ml−1 (after 3 days of incubation at 22°C) and 121 CFU ml−1 (after 15 days of incubation at 22°C) in the first loop of the experimental network (HRT 12 h). No massive biofilm sloughing was observed. The quality of nanofiltered water 2 changed during its transport through the network (total HRT of the water 72 h) with immediate bacterial contamination. But the total number of cells increased slowly and reached 1.4 × 105 cells ml−1 at the end of the system. The number of culturable bacteria decreased slowly and regularly as a function of the HRT to reach 8 CFU (3 days) ml−1 and 260 CFU (15 days) ml−1 at the end of the network.