The aim of this work was to characterize the sediment toxicity of a zone of the River Uruguay through a battery of bioassays. The samples were extracted from an area of influence of domestic wastewater discharges and pulp mill effluents (Fray Bentos), a touristic town downstream (Las Canas), and another one, upstream, isolated from those discharges (Nuevo Berlin). The bioassays: Photobacterium leiognathi toxicity screen, Ceriodaphnia dubia reproduction and embryo-larval teratogenicity and lethality with Pimephales promelas were applied to the sediment elutriates, while the Hyalella curvispina sub-lethal growth test was performed using whole sediment. To assess bioavailability and toxicity pathways, the sediment-water partitioning of 2,4,5-triclorofenol among phases was determined as: 63% dissolved, 27% suspended and the rest settled. Toxicity to organics and metals using P. leioghnati was observed in all sampled sites. The frequency of P. promelas vertebral malformations was 3.3% only to Fray Bentos elutriate, but the C. dubia reproduction bioassay showed effects only in those from Nuevo Berlin and Las Canas. The growth test with H. curvispina evidenced effects for all samples. The toxicity pathways could be ingestion of dissolved and particulate contaminants, possibly associated to higher clay and silt contents and contaminant input. The cause-effect relationships will be subject to further research.