The Hoosic River in northwestern Massachusetts drains into the Hudson River across from the Town of Stillwater, New York. Bacterial pollution is a continuing health concern in this region. While various efforts aim to monitor the levels of indicator bacteria (such as Escherichia coli and fecal coliform) in freshwater systems such as this, there has been relatively little research focused on determining how bacterial concentration fluctuates over different time scales throughout a watershed. Understanding regular spatial and temporal variation exhibited by populations of indicator organisms is necessary before robust water quality sampling programs can be put into place. This study examined the variation in E. coli at different time scales, including seasonal, diurnal, and weather-related in the upper Hoosic River Watershed. Based on research findings, several recommendations for improving water quality monitoring methods are offered.