Abstract: Understanding bacterial adsorption and the evolution of biofilms on arsenopyrite with different surface structures is of great significance to clarifying the mechanism of microbe–mineral interfacial interactions and the production of acidic mine drainage impacting the environment. In this study, the attachment of Sulfobacillus thermosulfidooxidans cells and subsequent biofilm formation on arsenopyrite with different surface structures in the presence of dissolved As(Ⅲ) was studied. Arsenopyrite slices with a specific surface were obtained by electrochemical corrosion at 0.26 V. The scanning electronic microscopy-energy dispersion spectra analyses indicated that the arsenopyrite surface deficient in sulfur and iron obtained by electrochemical treatment was not favorable for the initial adsorption of bacteria, and the addition of As(Ⅲ) inhibited the adsorption of microbial cells. Epifluorescence microscopy showed that the number of cells attaching to the arsenopyrite surface increased with time; however, biofilm formation was delayed significantly when As(Ⅲ) was added.