The objective of the present investigation has been to combine tracer principles and a hydrolytic microbial activity assay using fluorescein diacetate to monitor changes in microbial biomass within subsurface flow wetland mesocosms. The mesocosm hydrolytic activity was referenced to activated sludge concentrations treating a typical domestic wastewater at full scale. Microbial biomass activity levels within four laboratory wetland mesocosms treating a synthetic domestic wastewater were routinely monitored over a 21-week period of plant growth and rhizosphere development. Although above ground plant mass and tracer dispersion numbers suggested progressive root zone development, plant growth did not result in any measurable enhancement in microbial activity when compared to a mesocosm operating without plants. Dispersion numbers also suggested a reduction in the mass transport kinetics in these planted mesocosms. In-situ biomass monitoring enabled the assessment of a characteristic response in terms of the steady-state food to microorganism (F/M) ratio that was observed in mesocosms receiving both low and high organic loading. Wetland treatment performance is sensitive to the degree to which bed volume is exploited in terms of wastewater flow to regions of bioactivity. The in-situ reactive tracer technique for mesocosm biomass monitoring provided an assessment of the collective substratum and rhizosphere microbial biomass in direct contact with wastewater contaminants. Thus, in-situ biomass monitoring has application in further understanding of plant function and strategies for plant implementation in wetland research and development.