Studies have shown that fecal contamination can be determined by conducting multiple antibiotic resistance (MAR) analyses. The hypothesis is if bacteria exhibit resistance, they are likely to be derived from organisms exposed to antimicrobial agents. Therefore, this project seeks to apply MAR analysis to nonpoint source (NPS) and combined sewer overflow (CSO) areas along the Anacostia River in Washington, DC. Presumptive E. coli was isolated from NPS and CSO samples and tested with eight different antimicrobial agents to assess MAR indices. Isolates from CSO sources showed significantly greater resistance (p < .05) and higher MAR indices, with an average MAR index of 0.36 for CSO samples and 0.07 for NPS samples. It was also revealed that 96.9% of CSO isolates exhibited resistance, versus only 43.8% of NPS isolates. Our study on the Anacostia River using this approach clearly shows fecal coliforms are associated with CSO overflows, indicating that pollution-derived coliform levels are strongly linked to antimicrobial resistance. The implementation of this method as an index for water quality in the remediation of the Anacostia River has the ability to serve as a model and monitoring tool for the rehabilitation of urban watersheds.