Ports and harbors may represent a threat for coastal ecosystems due to pollutant inputs, especially those derived from maritime activities. In this study, we report a first assessment of the ecotoxicological threat posed by six ports and harbors of opposite coastal regions, Apulia and Albania, in the southern Adriatic Sea (Italy). A bioassay battery consisting of four different species representing different trophic levels, algae Dunaliella tertiolecta, bacteria Vibrio fischeri, crustacean Artemia salina, and echinoids Paracentrotus lividus, has been used to assess sediment elutriates, pore waters, and sediment suspensions. Two different approaches of toxicity data integration, worst case and integrated index, have been used to determine the most appropriate procedure for the investigated sites. All sites with the worst case approach showed high toxicity levels. The chronic test with algae was the most sensitive identifying the highest effects in the battery. This effect can be attributable to contaminants derived from antifouling paints. The sediments, evaluated with V. fischeri test, often showed toxicity not found in the aqueous matrices of the same sites and that can be mainly linked to organic compounds. The test battery used in this study allowed us to perform a preliminary screening of the ecotoxicological risk of the studied area. In fact, the species utilized for toxicity tests responded differently to the investigated samples, showing different sensitivity. The test battery integrated index did not allow highlighting the differences among the sites and showed a general high ecotoxicological risk. A larger number of tests with higher sensitivity together with a tailored attribution of weights to endpoints and matrices will improve the final site evaluation.