Unlike to natural organic matters (NOMs), effluent organic matters (EfOMs) are not well understood due to their complexity and heterogeneity. In this study, EfOMs from sewage and industrial wastewater treatment effluents and Suwannee River NOM (SRNOM) were isolated into hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) fractions. Specific ultraviolet absorbance (SUVA) and fluorescence excitation emission matrix (FEEM) analyses were used to characterize physicochemical properties. In addition, acute toxicity and oxidative stress to Daphnia magna were evaluated to characterize toxicological properties. EfOMs showed similar properties to microbially derived organic matters having low hydrophobicity, which are totally different from SRNOM having high hydrophobicity. Moreover, acute toxicity and antioxidant enzyme activity in D. magna was largely dependent on fraction types of EfOMs. These findings suggest that EfOMs have different physicochemical and toxicological properties compared with those of NOMs, which needs to be further identified with various sources of EfOMs.