Shipyards impact on estuarine environments because of the use of antifouling paints and petroleum products, which release trace metals that may remain in their bioavailable or labile form. Regardless of its importance, the relation between continuous input of trace metals (hotspot area) and their availability in the water column has been scarcely studied. This study evaluated seasonal variations in the concentrations of labile fractions of metals in shipyards located in estuarine areas on the Brazilian subtropical coast. These fractions were determined by the Diffuse Gradients in Thin Films (DGT) technique. Maximum labile fraction concentrations of Cr (0.3 μg L−1), Ni (2.2 μg L−1) and V (2.0 μg L−1) are directly related to (i) their specific source: antifouling paints (for Cr), metal and steel alloys (for Cr and Ni) and petroleum products (for V), besides (ii) periods of intensive traffic and vessel repair. Additionally, variations in labile fractions of Ni and V in the Patos Lagoon estuary were influenced by salinity, which is known to affect metal desorption from surface sediments in resuspension events. Even though Cr is affected by the same processes, it is available as Cr(III) and does not represent any ecological risk in the study areas. Although the areas under study are affected by variations in physical and chemical conditions, shipyards were effectively hotspots of trace metals in their labile fraction in various estuarine systems in southeastern and southern Brazil. Thus, they represent areas where Ecological Risk Assessment, mainly of V, should be carried out.