Along the coast of the State of São Paulo, Brazil, urban drainage channels introduce a complex mixture of pollutants into the South Atlantic Ocean, that may cause deleterious effects to the aquatic biota. The objective of this study was to analyse, for the first time, the mutagenicity (Ames Salmonella/microsome test) and ecotoxicity (acute and chronic tests, with Daphnia simillis and Ceriodaphnia dubia, respectively) exerted by the diffuse loads discharged in Guarujá, São Paulo coast, Brazil. Water sampling occurred bimonthly between January and July 2018 (rainy season: January through March; dry season: May through July) at four beaches with different profiles of use and land occupation: Tombo (Blue Flag certification), Enseada (high use by tourists), Perequê (fishing community) and Iporanga (conservation unit). No mutagenic potential was detected in the complex mixtures flowing to the study beaches. However, 30 and 80% of the analyses showed acute and chronic toxicities, respectively, mainly in the Enseada and Perequê channels during the rainy season. To improve the environmental quality of these coastal waters and to reduce the ecological risks posed to the aquatic organisms and public health, several actions are imperative, such as the amelioration of the basic sanitation facilities and land regularisation actions.