The vertical distribution of macrobenthic fauna, heavy metals, and other physico-chemical and biological characteristics of the sediments were studied in three sediment layers (0–7, 8–14, 15–21 cm) at seven stations in the Ubatuba region, north coast of São Paulo State, Brazil at several temporal and spatial scales. Six stations were located in the inner bay near the riverine run-off, and one was outside the bay, distant from the riverine influence. The samples were collected four times in 1 year, on a seasonal basis. Sediments were basis comprised predominantly of very fine sand and the vertical distribution of grain size was uniform to a depth of 21 cm in all stations. Higher values of total organic matter, organic carbon, sulphur, heavy metals and phaeopigments were recorded at the inner Ubatuba Bay stations, probably due to the riverine influence. C/N ratios indicated a mixed origin of organic matter with a major contribution of terrestrial material in the inner stations. The vertical distribution of heavy metals showed a slight decline with sediment depth in the inner stations, indicating the present contribution. Most of the macrofauna was found at the surface sediment layer. Biological data showed that in the inner stations of Ubatuba Bay, which are under the influence of urban sewage and are moderately polluted, the fauna was distributed more superficially within the substrate than in St. 7, which is located in the external portion of the bay distant from sewage inputs. The environmental quality of the sites studied varied little throughout the year, at least in relation to the variables considered here. Temporal variation in the vertical distribution of benthic fauna was not evident in the four sampling surveys analysed. Only minor changes in the vertical distribution of the total fauna were detected in the seasonal scale, with the organisms located less deep within the sediment column in summer, indicating some influence of the tourism impact and/or rainy season.