Graphene oxide (GO) sheets attracted great attention as effectively antibacterial agents in water treatment and environmental remediation applications. In the study, the interaction of humic acid (HA) as the model of natural organic matter (NOM) with GO and their antibacterial activities against Escherichia coli (E. coli) was investigated. The interaction between GO and HA molecules was analyzed by isothermal titration calorimetry (ITC) and fluorescence spectroscopy analysis. The study demonstrated that GO reaction with HA was a spontaneously exothermic process, which enabled formation of stable and well dispersed GO-HA complex in aqueous solution. Both GO and GO-HA could significantly inhibit the growth of E. coli and present dose-dependent bactericidal property. GO and GO-HA showed more obvious antibacterial activity in saline solution than in LB broth. We suggest the surface wrinkles of GO and GO-HA could contribute to the firm wrapping of E. coli, which is the principle factor for the antibacterial activity of GO and GO-HA. Especially, GO-HA exhibit less surface wrinkles in comparison with GO, corresponding to its reduced antibacterial activity in saline solution.