The present study evaluated the contamination of a surface water lagoon (Peri Lagoon) in Florianópolis, Santa Catarina, Brazil, by human adenovirus (HAdV), polyomavirus JC (JCPyV), hepatitis A virus (HAV) and rotavirus species A (RVA). Efforts were driven to determine the correlation between viral presence and the physicochemical parameters of the lagoon and measure the distribution of these viruses throughout the year (June 2010 to May 2011). A total of 48 samples were collected, concentrated and analyzed by qPCR (quantitative polymerase chain reaction). Approximately 96% of the samples were positive for HAdV (46/48), 65% were positive for RVA (31/48), 21% were positive for JCPyV (10/48) and 12% were positive for HAV (6/48). The presence of JCPyV was positively correlated with that of NO2−N, and also there was a positive correlation between the presence of each one of the viruses (HAdV, HAV and RVA) in winter. Samples from water dedicated for human consumption and recreation tested positive for HAdV by qPCR. These samples were also subjected to viral integrity and viability assays: 83% (10/12) contained intact viral particles and 66% (8/12) contained infectious particles. Our results demonstrate the release of human waste into water sources, justifying the urgent need to add viral parameters to water quality surveillance.