Harbor sediments normally accumulate the pollutants from municipal and industrial activities in the estuarine zone. This work aimed to characterize the fractionation change of heavy metals in harbor sediments before and after chemical washing. Since the annual dredging around the Kaohsiung Harbor has increased over time, the influence of ex-situ acid washing on sediment quality needs to be evaluated. Experimental parameters of acid washing included the solid loading (4–20%) and types (HCl, HNO3, and citric acid) and concentrations (0.01–1 M) of acids. The fractionation of Cu, Zn, Ni, Cd, Cr, and Pb in the sediments at three estuaries, the Chienchen River, Canon River (Dock No.5), and Yanshui River, before and after washing processes were determined through sequential extraction. The washing efficiencies of HCl, HNO3, and citric acid were similar, being 80.1–83.7%, 27.6–30.9%, 20.7–23.9%, and 97.2–98.8% for Zn, Ni, Cr, and Cd, respectively. After 15 min of washing, HCl was found as a suitable washing agent, and particularly, more effective for Cu (65.3%) and Pb (79.4%) than other acids. The extraction process suggested that the acid washing of carbonate, the Fe-Mn oxide composite, and organic phases were highly correlated to their quantities in the sediments. However, the removal efficiency was inversely related to the metals in the residual phase. Knowing the mobility and bioavailability of heavy metals based on fractionation of metals benefits the assessment of the potential risk of dredged harbor sediment after the washing procedure. This study provided evidence that acid washing, as a remediation method, could be versatile in removing heavy metals from mobile phases without causing mineralogical changes to the contaminated sediments of the harbor area.