Biosand filter (BSF) users are instructed to operate their filters with residence times of 1–48 hours. However, studies to date have only tested residence periods of 6–36 hours, with no studies extending to 48 hours, or investigating whether BSF performance decreases beyond 48 hours. The goal of this study was to compare Escherichia coli removal in filters operated with one-, two-, and three-day residence periods. Nine laboratory-scale column filters were operated in parallel, with three replicates for each of the three residence periods, over 84 days. Filters were fed with lake water supplemented with E. coli. Influent, effluent, and control samples were tested for a range of parameters including E. coli colony forming units; turbidity; pH; electrical conductivity; dissolved oxygen (DO); and nitrogen as ammonia, nitrate, and nitrite. Hydraulic conductivity and DO of pore water were measured for different sand depths within the filters every six days. The study found no significant difference in E. coli removal by extending the residence period to three days. However, water treated in filters with increased residence periods had lower DO concentrations and increased nitrite levels.