Chemical functionalization is widely adopted to increase the hydrophilicity of fullerene for various applications. Protein–nanomaterial (NM) interactions are one of the very first issues after NMs enter biological systems, which determines their bio-effects and toxicity. To better understand the biological and environmental effects of fullerene, we investigated the influence of surface modification on the fullerene–protein interaction by experimental and computational approaches. Pristine fullerene (C₆₀) was functionalized to obtain esterified fullerene (C₆₀–COOR), carboxylated fullerene (C₆₀–COOH) and fullerenol (C₆₀–OH), resulting in higher hydrophilicity and dispersibility following the sequence C₆₀–OH > C₆₀–COOH > C₆₀–COOR > C₆₀. The docking results suggested that good hydrophilic functionalization largely reduced the π–π interaction between aromatic residues and the fullerene cage, resulting in a weaker interaction between fullerene and lysozyme. Experimentally, the chemically functionalized fullerene showed more inhibition of enzyme activity. Correspondingly, the protein conformation changed more after incubation with functionalized fullerene with hydrophilic moieties (C₆₀–COOH and C₆₀–OH), indicated by the increased UV absorbance, the inhibition of intrinsic fluorescence and the loss of the α-helix structure. The contradictory results of computational and experimental approaches were attributed to the different dispersibilities, where functionalized fullerene with hydrophilic moieties dispersed well in aqueous systems to reach the lysozyme active pocket more easily. As a direct bio-effect, the dispersible fullerenes inhibited the antibacterial activity of lysozyme, which might affect the resistance to infection and the applications of lysozyme in biomedicine, food and bioengineering. Our findings suggested that surface modification should be carefully designed to obtain safer carbon NMs and enough consideration should be paid to the protein–nanomaterial interaction in environmental toxicity evaluations.