Although Tween-20 was used as an important catalyst to increase chalcopyrite bioleaching rate by acidophiles, the effect of Tween-20 on initial adhesion and biofilm development of acidophiles on chalcopyrite has not been explored until now. Herein, the role of Tween-20 in early attachment behaviors and biofilm development by Acidianus manzaensis strain YN-25 were investigated by adhesion experiments, adhesion force measurement, visualization of biofilm assays and a series of analyses including extended Derjaguin Landau Verwey Overbeek (DLVO) theory, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The bacterial adhesion experiments showed that 2 mg/L of Tween-20 increased the adhesion percentage (by 8%) of A. manzaensis YN-25. Tween-20 could promote the early adhesion of A. manzaensis YN-25 by changing the Lewis acid-base interaction and electrostatic force to increase total interaction energy and adhesion force. Besides, the functional groups on the surface of cells (carboxyl, hydroxyl and amino functional groups) contributed to the adhesion of A. manzaensis YN-25 on chalcopyrite. Furthermore, the promotion of biofilm formation by Tween-20 was mainly attributed to the reduction of S0 passivation layer formation and complexing more Fe3+ on chalcopyrite surface, contributing to the erosion of chalcopyrite and creating more corrosion pits. Live/dead staining showed low live/dead ratio (ranged from 0.35 to 1.32) during the biofilm development process. This report offers a better understanding of the effects of Tween-20 on attachment and biofilm development of acidophilic microorganisms and would lay a theoretical foundation for the better application of catalyst in bioleaching.