The proliferation of petrochemical plants close to natural water bodies has resulted in emission of numerous organic pollutants into aquatic environments. However, the molecular composition of petrochemical effluent organic matter (PEfOM) is poorly understood, which results in uncertainty regarding its environmental effects and potential risks. In this study, optical spectroscopy and mass spectrometry coupled with both negative-ion ((−)ESI mode) and positive-ion electrospray ionization mode ((+)ESI mode) were used to conduct large-scale molecular-level characterization of PEfOM from 21 petrochemical plants in China. In contrast to natural organic matter (NOM), PEfOM was characterized by low aromaticity and high microbial-derived fluorescence. These properties corresponded to a composition rich in aliphatic and highly unsaturated phenolic compounds, as well as low average molecular weight and elevated heteroatom content. Among the 21 PEfOM samples, molecular compositions exhibited significant heterogeneity with Jensen–Shannon divergence values between 0 and 0.2. Based on the O/S/N contents, aromaticity, and unsaturation, the compounds in PEfOM were grouped into three clusters under (+)ESI mode (+CL1, +CL2, +CL3) and two clusters under (−)ESI mode (−CL1 and −CL2). Nitrogen-rich CHON compounds with high double bond equivalents (4–14) in +CL2 and oxygen-poor compounds with high molecular weights (600–800 Da) in −CL1 exhibited the greatest reactivity in aquatic environments under aerobic conditions. The compounds in these (+)ESI and (−)ESI clusters had Gibbs's free energy of carbon oxidation values of 80–110 and 60–100, respectively. C17H23N1O2 (quinolinium derivatives), C14H20O5S1 (benzene sulfonic acid), and C9H17N1O5S1 (amino-sulfonates) were identified as characteristic pollutants with high indicator species values. Ecological structure activity relationships analysis revealed that C17H23N1O2 exhibited high acute biotoxicity, which was associated with potential environmental risks. This study offers valuable insight and guidance for risk management and control of petrochemical effluents.