The microbiological pollution of coastal waters is a major problem, especially in shellfish areas. This article shows the faecal contamination in bivalves from the Ria Formosa Lagoon (south coast of Portugal) along 20 years (1990-2009). The highest values of Escherichia coli in bivalves were obtained during the 90s, related with the discharge of untreated wastewaters and agricultural runoff. In the 2000s contamination levels decreased, with 83% of the population already served by new or remodelled sewage treatment plants. The highest levels were found in bivalves close to the largest city, where punctual and diffuse contamination sources still exist. Bivalves from the less impacted site showed the lowest contamination, an area with more water renewal. Seasonally, the highest levels were in autumn and winter, due to the runoff of waters from rainfall. These were opposite to those in spring and summer, when the highest temperatures and salinity showed a bactericidal effect.