Soil treatment units (STUs) receiving effluent from on-site wastewater treatment systems (OWTSs) rely on the gradual development of a microbial biomat/biozone at the infiltrative surface for optimal effluent distribution and pollutant attenuation. Here, we present the first direct measurement of gradual biomat development in the field in STU trenches receiving either primary (PE) or secondary treated effluent (SE) under identical environmental, hydrological and subsoil conditions. Two domestic OWTSs were constructed in Ireland and monitored over a period of >2 years using an automated, three-dimensional network of buried soil water content sensors tracking water flow and retention within the soil underneath the infiltrative surface. While trenches receiving PE expressed signs of biomat formation along the entire length of STU trenches, biomats in trenches receiving SE were significantly muted and did not extend further than 10 m from the inlet at the end of the study. The presence of a mature biomat helped to retain soil moisture above background levels and made the system more resilient towards drought events and desiccation stress but led, in one case, to effluent ponding within the trenches. A growth-limited non-linear model fit revealed that biomats in SE trenches are expected to remain considerably shorter and will not spread along the entire trench design length, even after 10 years of operation, which is contrary to prevalent design assumptions. Muted biomat growth, on the contrary, might lead to localized hydraulic and pollutant overloading and has been shown previously to negatively affect the ability to attenuate pollutants effectively within the soil profile before the effluent reaches the groundwater.Copyright © 2020 Elsevier B.V. All rights reserved.