To evaluate the hazard assessment of dredging and disposal of sediments, an in-depth chemical and toxicological characterization of sediments was carried out in Venice Lagoon. The bioaccessible and mobile concentration of trace elements in superficial and deep sediments (in the Holocene-Pleistocene limit) can deeply affect the quality of the aquatic ecosystem. Geospeciation and total concentration of trace elements in sediment cores were assessed and several toxicological tests were carried out. The indices of contamination (e.g., geochemical accumulation index, Igeo, pollution load index, PLI) and of speciation (individual contamination factor, ICF and global contamination factor, GCF) were evaluated. Factor Analysis (FA) was performed to explore possible significant correlations between toxicity data and sediment chemistry. Results underlined that the total concentration of trace elements cannot adequately assess the hazard, while bioavailability, mobility, and toxicity of trace elements allow a thorough evaluation of the environmental threats. The integration of results from chemical characterization ex ante and toxicity assays will provide for a better sustainable handling and management of sediments.