Microbial contamination is now more common than chemical contamination in Tibet, and water-borne microbes can cause a number of diseases that threaten public health. Thus, in order to clarify the spatiotemporal distribution of bacteria in small watersheds for which there is no data in Tibet, we set up four sampling points along an upstream-downstream transect of the Xincang River Basin. We collected 239 water samples in 2014 and 2015, and evaluated their total constituent numbers of bacteria (TB) and coliforms (TC). The results of this study show that the microbial contamination of the Xincang River Basin is mild-to-moderate in terms of TB and TC contents, and that these concentrations vary significantly in different seasons. Results show that in summer TB and TC concentrations in the downstream section of this river were highest and that microbial contamination was most serious. Data also demonstrate that precipitation is the most important factor underlying increases in TB and TC concentrations during the summer months; both these variables are significantly correlated with precipitation, while animal husbandry and domestic sewage are the main sources of microbial contamination overall. The results of this study are likely to reflect the basic characteristics of small watersheds for which there is no data to some extent, and are thus of significant practical importance for protecting their ecological environments and promoting sustainable development.