The aim of this study was to evaluate the water quality of the port of Otranto (Italy), through a combined approach which integrates monitoring of traditional microbiological parameters and studying the dynamics of some autochthonous bacterial communities. The fecal contamination indicators, total coliforms, fecal coliforms, and fecal streptococci were measured to evaluate the sanitary aspects, while the presence of organic matter was considered as a parameter strictly related to dumping. In addition, being the port of Otranto especially exposed to hydrocarbon pollution, hydrocarbon-oxidizing bacteria were evaluated. Fecal contamination indicators were consistently found to be below the threshold values set by the Italian legislative decree N. 470/82 for bathing waters, indicating a good microbiological quality of these waters. A higher density of hydrocarbon-oxidizing bacteria was found at mooring and craft transit areas. This parameter was therefore found to be useful for evaluating port water hydrocarbon pollution.