Aims To evaluate the efficacy of using in situ diffusion chambers for pathogen decay studies in the aquifer. Methods and Results A comparative microbial inactivation study was carried out in groundwater by seeding selected pathogens and indicators in laboratory microcosms and Teflon diffusion chambers (in situ) fitted with 0.010- and 0.025-mu m pore-size membranes. The results have shown that there is a difference in the decay rates obtained from the laboratory microcosms and in situ diffusion chambers for several pathogens. The results suggest that the use of laboratory microcosms to determine pathogen decay during the Managed Aquifer Recharge (MAR) may lead to underestimation of decay of adenovirus and Cryptosporidium and subsequent inaccurate assessment of the required residence time in the aquifer for pathogens to be removed from the recharged water. The decay rate of two indicator bacteria, Salmonella enterica and adenovirus, was found to be significantly slower (t-test, P < 0.05) in the in situ studies when 0.010-mu m pore-size membranes were used. The assessment of potential clogging of the diffusion cell membranes showed no clogging of the membranes within the 50-day duration of the study as very little difference in the diffusion rates between new and old membranes was observed. Conclusions Laboratory microcosms-based inactivation studies can significantly underestimate the survival potential of enteric viruses in the groundwater. Consequently, in situ studies should be carried out for accurate assessment of potential enteric virus survival in aquifers. Significance and Impact of the Study Reliable assessment of potential public health risks from the presence of pathogens in groundwater is essential for proper management of the MAR schemes. The results of this study suggest that in situ assessment of pathogen survival potential in diffusion chambers provided more reliable data for pathogen risk assessment and subsequent risk mitigation plans for MAR schemes.