The current work tackles the problem of soil microbial growth and biofilm expansion assessment in infiltration percolation process in order to optimize the technique design and monitoring. Experiments were conducted on a sand filled PVC column fed at a pulsed rhythm of 8 sequences per day in order to maximize the soil aeration that generates a better oxygen renewal in the soil gas phase, enhances the oxidation of the oxidisable matters and improves sanitary performances. As we target the biofilm expansion study, we assessed the microbial biomass at different depths of the soil matrix (10, 25, 50 and 75 cm), by the chloroform fumigation-extraction and DNA extraction methods. Results showed that the microbial biomass increases with operating time especially between 10 and 25 cm depths until the 96(th) operating day (OPD) and decreases notably after this time. A highly significant positively correlation (r = 0.97) is registered between results of microbial biomass quantification by fumigation-extraction method (FEM) expressed as carbon material (biomass C) and those obtained by DNA extraction method (DNA-EM). In addition, we performed an enumeration of the biofilm total microbial flora using the solid medium cultivation method which corroborates the microbial biomass quantification results. In order to illustrate the biofilm shape generated by bacterial cells and extracellular polymeric substances (EPS) in the vicinity of the sand grains, we performed different scanning electron microscope (SEM) micrographs at different elapsed operation times.