Tropical estuaries are threatened by rapid urbanization, which leads to the spread of thousands of micropollutants and poses an environmental risk to such sensitive aqueous ecosystems. In the present study, a combination of chemical and bioanalytical water characterization was applied to investigate the impact of Ho Chi Minh megacity (HCMC, 9.2 million inhabitants in 2021) on the Saigon River and its estuary and provide a comprehensive water quality assessment. Water samples were collected along a 140-km stretch integrating the river-estuary continuum from upstream HCMC down to the estuary mouth in the East Sea. Additional water samples were collected at the mouth of the four main canals of the city center. Chemical analysis was performed targeting up to 217 micropollutants (pharmaceuticals, plasticizers, PFASs, flame retardants, hormones, pesticides). Bioanalysis was performed using six in-vitro bioassays for hormone receptor-mediated effects, xenobiotic metabolism pathways and oxidative stress response, respectively, all accompanied by cytotoxicity measurement. A total of 120 micropollutants were detected and displayed high variability along the river continuum with total concentration ranging from 0.25 to 78 μg L−1. Among them, 59 micropollutants were ubiquitous (detection frequency ≥ 80 %). An attenuation was observed in concentration and effect profiles towards the estuary. The urban canals were identified as major sources of micropollutants and bioactivity to the river, and one canal (Bến Nghé) exceeded the effect-based trigger values derived for estrogenicity and xenobiotic metabolism. Iceberg modelling apportioned the contribution of the quantified and the unknown chemicals to the measured effects. Diuron, metolachlor, chlorpyrifos, daidzein, genistein, climbazole, mebendazole and telmisartan were identified as main risk drivers of the oxidative stress response and xenobiotic metabolism pathway activation. Our study reinforced the need for improved wastewater management and deeper evaluations of the occurrence and fate of micropollutants in urbanized tropical estuarine environments.