Cross-border flow of untreated sewage and pollution through the Tijuana River Estuary (TRE) have been a public health hazard and source of contention between the United States and Mexico for decades. Pollution problems have been exacerbated by rapid population growth, straining Tijuana’s aging and undersized sewage infrastructure resulting in frequent untreated sewage spills into the Estuary. In this comprehensive environmental and public health risk assesment at Tijuana River Estuary, we quantified pollutants in estuarine water and sediment, and are investigating the effects of both pathogens and chemical pollutants on human and ecosystem health utilizing environmental toxicology and metagenomics analyses. Our goal is to assist in ongoing efforts to develop both real-time and long-term broad testing/monitoring programs resulting in effective mitigation strategies to minimize the human and ecological risk associated with the Tijuana River Estuary, providing stakeholders with the knowledge to prioritize, assess and manage these risks.