This work aimed to study the short, medium, and long-term influences of treated wastewater (WWTP) as a soil irrigation technique on physical, chemical, and microbiological properties. Sampled soil plots were irrigated with WWTP for 1, 7, and 15 years. Rain-fed regime soil has been used as a control. This study was conducted in El Hamma of Gabes (southeast of Tunisia), an arid zone, during the dry season. Three soil profiles at 0–20, 20–40, and 40–60 cm were sampled. Physical and chemical analyzes, including pH, electrical conductivity, total organic carbon, total nitrogen, cation exchange capacity, and active and total CaCO3 were evaluated. Soil microbial biomass C and N were performed according to the fumigation extraction method. Soil microbial contamination was assessed by enumerating total, Fecal Coliforms, E. coli, and Fecal Streptococci with the most probable number (MPN) method and plate medium count. Bacterial and fungal enumeration were evaluated by the plate count agar method. In addition, the DGGE molecular analysis was also assessed in the different soil samples. The obtained physical and chemical evaluation proved that the WWTP irrigation practice improves soil fertility. Microbial biomass C and N increased by increasing the time of WWTP irrigation. In addition, the sanitary quality of the soil is affected by the increase of pathogens. We have observed an increase in microbial biomass as demonstrated by an increase in heterotrophic bacteria and filamentous fungi in the soil irrigated with WWTP compared to the control. Long-term WWTP irrigation has increased the activities of the soil microorganisms and then soil fertility. Meanwhile, the same pathogens and bacteria could pose a serious threat to sanitary soil quality and a limiting factor for the use of WWTP in agriculture areas without tertiary treatment.