Biofilms in drinking water network are ubiquitous, hosting bacteria that must be assessed and controlled, particularly pathogens. Bacterial population can be affected by operational conditions in the network. Variables including distance, time of stabilization, velocity and high (biofilm 1) and low (biofilm 2) chlorine were assessed as modifiers of bacterial population, in a pilot-scale system during a long-term period and laminar conditions. Gram-positive bacteria and other Proteobacteria, pathogens and versatile bacteria were quantified in biofilm samples by fluorescent in situ hybridization (FISH), using respectively Eubacteria 338I, Gamma- and Betaproteobacteria probes. Denaturing Gradient Gel Electrophoresis (DGGE) analyses were also performed to determine similarity between samples. Based on results, a high heterogeneity was observed even at short length (3 m: <40% similarity). The time of stabilization changed the effect of velocity over bacterial population. After 169 days, water velocity was negatively correlated with Betaproteobacteria (r = −0.71; p = 0.11 in biofilm 1) and Eubacteria 338I (r = −0.91; p = 0.01 in biofilm 2), while at the end, Eubacteria 338I from biofilm 2 correlated positively with water velocity (r = 0.78; p = 0.06). Chlorine favored the proliferation of Gamma-, Betaproteobacteria, especially in biofilm 1, being Gammaproteobacteria counts one order higher than in biofilm 2. Chlorine proved to be the most influential operational condition on the bacterial population, because it benefited the group characterized by pathogenicity. Due to the scarce information about mature biofilms at low flux conditions, this study contributes to the understanding on Eubacteria 338I, Gamma- and Betaproteobacteria populations, thus to water distribution system management in rural operation.