The present work aims to ascertain the mechanisms of surfactant (dodecylbenzene sulfonate; DBS) effects on the aggregation behaviors of TiO2 nanoparticles (TiO2-NPs) in natural water samples. Aggregation experiments were conducted at a TiO2-NPs concentration of 10mg/L in deionized water and in natural water samples via dynamic light scattering and Zeta potential determination. Average attachment efficiency was calculated to compare the aggregation behaviors of nanoparticles in the two aqueous media. Results showed that the effects of DBS on aggregation could be interpreted by both Derjaguin–Landau–Verwey–Overbeek (DLVO) and non-DLVO mechanisms. In natural water samples, aggregation did not occur rapidly and was able to develop slowly under all conditions, and the roles of DBS were obvious at high DBS concentration owing to the impacts of inherent components of natural water samples, such as colloids and natural organic compounds. Future aggregation studies should concentrate on multi-factor, multi-colloidal and dynamic aspects under similar environmental conditions.