In on-site wastewater treatment plants, effluents are pre-treated by septic tank and treated by soil infiltration or sand filtration systems, with unsaturated flow conditions. These systems remove efficiently carbon, nitrogen and suspended solids. But for microbial pollution, the treatment efficiency depends on the hydrodynamic behaviour and filtering media characteristics. Contamination of superficial water and groundwater due to pathogenic viruses and pathogenic bacteria is responsible for many diseases. The objective of this study is to approach the mechanisms and operating conditions to control bacteria and virus release in the environment. Experiments were carried out on reactors of different length packed with sand. Hydraulic load of 90 cm x d(-1) with a pulse periodic flow was used. The influence of chemical composition of the solution on the treatment efficiency has also been studied. For the first time, the residence time distribution (RTD) has been studied using a conservative tracer (KI), to determine the main hydrodynamic parameters. For the second time, the RTD with bacterial and viral tracers (E. coli, bacteriophage MS2) was applied, with the aim to define microbial behaviour in filtering media, including adsorption and filtration phenomena. This work allowed us to determine retardation factors according to the hydraulic loads and chemical composition.