In Southern California, storm events have resulted in regular exceedances of water quality benchmarks in rivers. Some of the suspected sources of anthropogenic contamination in urban waterbodies are sanitary sewer overflows (SSOs), illegal connections and discharges, and wastes from homeless encampments. Temporal changes in surface water level and flood volume also have the potential to inundate more contaminant source areas and increase hydraulic connectivity between groundwater, vadose zone, and surface water, thereby increasing the sources or magnitude of contaminants over the duration of a storm. The aim of this study was to analyze the possible sources of contamination by evaluating the temporal trends in loadings and concentrations of biological and chemical constituents during storm events. We also looked at the chemical and biological characteristics of different end- member environments (water matrices) to distinguish the different sources of pollution to the San Diego River, and its tributaries. Stormwater samples were collected during five storm events spanning a range of intensity and duration during the 2018 and 2019 hydrologic years. Traditional fecal indicator bacteria (FIB), microbial source tracking (MST) markers (i.e., HF 183 and Pepper Mild Mottle Virus (PMMoV)), caffeine, sucralose, and optical spectroscopic properties were monitored as tracers of human contamination. Multiple lines of evidence revealed that, with the exception of one rain event for which the precipitation was too low to produce interflow, the main source of microbial pollution to the San Diego River in four out of the five storm events sampled was untreated wastewater mobilized from the subsurface through interflow. This study demonstrated that the use of caffeine/sucralose ratios and the human-associated marker, HF183, hold promise for tracking sewage inputs. In addition, changes in the chloride/bromide ratio showed a transition from saline groundwater inputs at baseflow to rainfed interflow during storms, and changes in specific UV absorbance and fluorescence index helped discern the flushing of soil pore water from contributions of surface runoff during storm events. The findings of this study have important implications for stormwater management and highlight the need for maintenance and repair of aging sewer infrastructure.