||The industrial-scale fullerene production has steadily increased since 1990 and reached at least 10 tons in 2007. This implies that a substantial amount of C60 may end up in aquatic environments. The negatively charged aggregates of C60 (nC60) are found to be stable in the aqueous environment, which has elicited a growing concerns over the potential environmental effects and health risks of these aggregates. More and more studies have thus been conducted to investigate the negative impacts of C60 on environment and human health. However, there has still been a debate about potential toxicity of C60 for over a decade because different preparation methods change physicochemical properties of the fullerene aggregates (nC60) in terms of sizes, shapes, chemistry, crystallinity, surface properties, and agglomeration status, thus leading to contradictory conclusions on fullerene toxicity. This research is therefore aimed at investigating the impacts of preparation methods on physicochemical properties and the effect of the key property parameters on the toxicity to E. coli. Four typical methods for preparation of nC60 suspensions were adopted in this study, including extended mixing in water (with or without oxygen), solvent exchange with toluene or tetrahydrofuran (THF). The resulted four types of nC60 containing suspensions are named Aqu-N2/nC60, Aqu-O2/nC60, Tol/nC60 and THF/nC60, respectively, in which Aqu-N2/nC60 is taken into consideration for the first time. With the appropriate control, it can be achieved four types on nC60 suspensions in the same range of particle size with similar agglomeration status and crystallinity. However, the aggregates morphologies and surface chemical modifications were varied from each other. In summary, the dispersion and agglomeration happened spontaneously during the preparation in all types of nC60 aggregates, in which the surface modification is an important factor especially when the mechanical force is weak. Based on these resulted nC60 aggregates, they were spiked to nutrient broth for pure culture of Escherichia coli (E. coli AMC 198) growth at different levels of each nC60. Inhibition and lethality were observed in the presence of THF/nC60, which increased with THF/nC60 dose. THF/nC60 was identified to be very toxic with an EC50 at 0.54 mg/l. The other three types of nC60 aggregates have no clearly effects on E. coli. However, the genotoxicity study has discovered that all nC60 suspensions damaged the membrane, protein and DNA of the bacteria when fullerene concentrations were as high as 1 mg/l. The potential toxic mechanism of THF/nC60 has been evaluated based on the studies of physicochemical properties, biosorption and ROS generation, which could be as follows: the biosorption and ROS generation characteristics can affect the toxicity of nC60 aggregates, and the biosorption of nC60 onto E. coli can further be affected by solvent residual and surface modification in the aggregates; ROS generation can be influenced with crystal structure and surface modification of the aggregates. THF/nC60 surface tends to be less chemically modified during the dispersal process so that they can be easily adsorbed/absorbed onto/into bacteria, during which they produce large amount of cytotoxic singlet oxygen. Therefore, THF/nC60 is more toxic than the other three types of nC60 aggregates.