<p>The Santiago-Guadalajara River, located in western Mexico, is one of the most polluted rivers in the country, with widespread fecal contamination that presents a potential risk to public health, food safety, and biodiversity. This study investigated the spatial and seasonal behavior of fecal indicator bacteria (FIB) in the Santiago-Guadalajara River Basin (SGRB) using the most probable number (MPN) microbiological quantification technique and measuring the tributary flow rates in the main stem of the river and tributaries. Twenty-five sampling sites were monitored from July 2021 to April 2022. The mean of the microbial counts (MPN/100&#xa0;mL) at the basin was as follows: total coliforms 2.5 × 10⁷, fecal coliforms 2.2 × 10⁷, and <i>E. coli</i> 2.1 × 10⁷. These FIB values position the Santiago-Guadalajara River as one of the most polluted rivers globally since it significantly exceeds the regulatory limits at the monitored sites, indicating insufficient sanitation infrastructure throughout the basin. A high level of correlation was found between FIB concentrations and monitored stream tributary flows, which allowed modeling the behavior of the FIB with respect to the flow regime throughout the basin. Quantitative microbial risk assessment revealed specific stations with elevated infection risks from <i>Escherichia coli</i> exposure. This field-based study provides valuable insights into the relationship between the variables that influence FIB concentrations in a highly polluted river and the potential risk to the exposed population.</p>