Environmental pollution from synthetic chemical mixtures has significant adverse impacts on marine ecosystems. However, identifying the main constituents of chemical mixtures that pose ecological threats is challenging due to the necessity of an integrated workflow for comprehensive identification and toxicological prioritization of pollutants. Here, an all-in-one mass spectrometric strategy integrating target, suspect, and nontarget analysis was used to investigate organic pollutants of concern in fishing port sediments, with 355 pollutants (32 from target analysis, 118 from suspect screening and 205 from nontarget analysis) identified in 11 categories. The chemical classes of polycyclic aromatic hydrocarbons (PAHs), pesticides, and intermediates were the extensively detected chemical classes. The ecological risks of absolutely quantified pollutants (i.e., 16 parent PAHs, 7 organophosphate esters (OPEs), 10 pesticides and 4 benzotriazole ultraviolet absorbers) were assessed using toxicity-weighted concentration ranking, with o,p'-DDT being the major contributor. Under the toxicological priority index (ToxPi) framework, an extended ranking of all identified pollutants was achieved by combining instrument response and detection frequency, with a priority control list of 15 pollutants obtained, of which benzo[ghi]perylene (BghiP) and p,p'-DDE had the highest risk priority. Due to frequent detection rates and significant environmental risks, routine monitoring of petroleum pollutants is considered essential. This study presents a general workflow that includes comprehensive identification and prioritization of pollutants, facilitating chemical management and ecological risk assessment.