Because of their significant impact on public health, waterborne pathogens, especially bacteria and viruses, are frequently monitored in surface water to assess microbial quality of water bodies. However, more than one billion people worldwide currently lack access to safe drinking water, and a diversity of waterborne outbreaks caused by pathogens is reported in nations at all levels of economic development. Spatio-temporal distribution of conventional pollutants and five pathogenic microorganisms were discussed for the Haihe River Basin. Land use and socio-economic assessments were coupled with comprehensive water quality monitoring. Physical, chemical, and biological parameters were measured at 20 different sites in the watershed for 1 year, including pH, temperature, conductivity, dissolved oxygen, turbidity, chemical oxygen demand, ammonia-N, total and fecal coliforms, E. coli, and Enterococcus. The results highlighted the high spatio-temporal variability in pathogen distribution at watershed scale: high concentration of somatic coliphages and fecal indicator bacteria in March and December and their very low concentration in June and September. All pathogens were positively correlated to urban/rural residential/industrial land and negatively correlated to other four land use types. Microbial pollution was greatly correlated with population density, urbanization rate, and percentage of the tertiary industry in the gross domestic product. In the future, river microbial risk control strategy should focus more on the effective management of secondary effluent of wastewater treatment plant and land around rivers.