Industrial effluents, agricultural runoff, and municipal wastewaters contain unknown substances and complex mixtures that are released into the environment and can lead to contamination of surface and subsurface waters. In the present report, we have used the alkaline Comet assay and the micronucleus (MN) test to detect the genotoxicity due to multiple sources of pollution in the peripheral blood of two native estuarine fish (mullet and sea catfish) and evaluated possible interactive genotoxic effects from multiple contaminants and the seasonal variation of the genotoxicity. Mullet and sea catfish were captured in the Tramandai and Mampituba Rivers in the southern Brazilian state of Rio Grande do Sul. Reference animals were obtained from the Armazem lagoon. Fish captured in the two estuaries during the four seasons over a period of 2 years had increased levels of DNA damage and MN frequencies relative to the reference fish. In general, the alkaline Comet assay was more sensitive to the genotoxicity of the river contaminants than the MN test. The Comet assay demonstrated significant differences in fish captured at different seasons and at the two river sites, while the MN test showed significant differences only for the annual average for mullet from both sites and fish from the control site. The increases in DNA damage appear to be related to the increase in the number of people in the towns close to the study areas during the warm spring and summer seasons. Although no specific cause-effect relationships were established, comparison of the chemical contaminants and physical variations in the rivers with the genotoxicity data indicate that there may be some association between hydrocarbons, metals, pH, and water temperature and the level of damaged cells observed in mullet and sea catfish from the Tramandai and Mampituba estuaries.