The purpose of our investigation was to assess the water quality around the harbour of Otranto (Lecce, Italy) through an approach that integrates the determination of the classic parameters within traditional microbiological monitoring (the faecal contamination indexes—total coliforms, faecal coliforms, and faecal streptococcus) with the study of autochthon microbiological populations dynamics (hydrocarbon-degrading bacteria and bioluminescent bacteria). The analysis of our data shows that the faecal contamination indexes have values below the limits fixed by the 76/160/EEC directive for the bathing waters, marking the good microbiological quality of these waters. The density of the hydrocarbon-degrading bacteria has been shown to be much higher in the areas designated for the ships' berthing and for the transit of the crafts; therefore, this parameter has shown itself to be useful in the assessment of hydrocarbon contamination in harbour areas. The bioluminescent component of the epibacteria community was set as a marker for the biological quality of the seawater: the relative abundance of the bioluminescent bacteria in the impacted sites is below 5%.