The characteristics of effluent organic matter (EfOM) and the type of disinfection methods are closely related to the formation of disinfection byproducts (DBPs) in reclaimed water. In this study, five disinfection methods, i.e., chlorination, ultraviolet (UV) followed by chlorination (UV + Cl), UV/chlorine (UV/Cl), chloramination, and chlorine dioxide (ClO₂), were applied to investigate the changes in the properties of EfOM, the formation of DBPs, and the relationship between EfOM properties and DBP formation during the disinfection of four secondary biological effluents. The results showed that EfOM with medium molecular weight (MW) (0.5–6 kDa) was the dominant fraction for all WWTPs. From a fluorescence perspective, the EfOM of the AAO process was rich in humic matter, while the EfOM of the oxidation ditch (OD) process was rich in protein matter. Disinfectants tended to transfer EfOM with high molecular weight (MW) (>6 kDa) to those with low MW (<0.5 kDa). Chlorination, UV + Cl and UV/Cl were more reactive to humic matter, while chloramination and chlorine dioxide were more reactive to protein matter. The formation of known DBPs was mainly dependent on humic matter, while protein matter was more likely to generate unknown DBPs. N-DBPs only accounted for 5.7%–17.7% of the total DBPs, but contributed more than 70% of the calculated toxicity, among which bromochloroacetonitrile (BCAN), dichloroacetonitrile (DCAN), and monobromoacetamide (MBAcAm) were the most important contributors to the calculated cytotoxicity. Monobromoacetic acid (MBAA) and MBAcAm were the primary drivers of the calculated genotoxicity. Overall, UV + Cl was the suggested optimal disinfection method for WWTPs.