Water quality monitoring and microbial risk assessment are important to ensure safe water for drinking, recreational, and agricultural purposes. In this study, we applied a microfluidic quantitative PCR (MFQPCR) approach to simultaneously quantify multiple waterborne pathogens in a natural freshwater lake in Hokkaido, Japan, from April to November, 2012. Tens of thousands of geese stopped over at this lake during their migration in spring and fall. Because lake water is used for irrigation of the surrounding agricultural area, we assessed infection risks through irrigation water usage based on pathogen concentrations directly measured by MFQPCR. We detected various pathogens in the lake water, particularly during the bird migration seasons, suggesting that migratory birds were the main source of the pathogens. However, neither counts of geese nor fecal indicator bacteria were good predictors of pathogen concentrations. On the basis of quantitative microbial risk assessment, concentrations of Campylobacter jejuni and Shigella spp. in water samples were above the concentrations that can potentially cause 10(-4) infections per person per year when water is used to grow fresh vegetables. These results suggest that direct and simultaneous multipathogen quantification can provide more reliable and comprehensive information for risk assessment than the current fecal indicator-based approach.