To develop effective groundwater pollution control strategies for the Kathmandu Valley, Nepal, seasonal variations in microbial quality and their underlying mechanisms must be understood. However, to date, there are no studies that address these topics. In this study, groundwater samples from dug wells were collected during the dry and wet seasons from 2009 to 2012, and Escherichia coli (E. coli) and total coliforms were analysed. Three wells were monitored each month for a year. Microbial concentrations in shallow groundwater were significantly higher during the wet season than during the dry season. Analyses of rainfall and E. coli concentrations in different seasons indicated that a high level of faecal material infiltration during the rainy season may have caused the seasonal variations in microbial quality. A moderate to strong relationship between E. coli concentrations and groundwater level suggested that the rise in groundwater levels during the wet season may be another reason for this variation. This long time-scale survey detected a significant decline in the microbial quality of shallow groundwater during the wet season as compared with the dry season. We propose that the infiltration of contaminants and change in groundwater level are the two probable mechanisms for the observed seasonal differences.