Abstract: Jordisite (MoS₂) is one of the most difficult minerals to deal with in the development of molybdenum-containing mineral resources because of its characteristics of fine molybdenum content, low liberation degree, easiness of slime formation, and poor floatability. Much attention has been paid to develop an effective, economical, and eco-friendly processing method. In this study, a nonionic surfactant Tween 20 was used to improve the leaching of jordisite in the presence of Acidithiobacillus ferrooxidans. The effects of Tween 20 on the metabolic activity of A. ferrooxidans and on the bioleaching of jordisite were tested. After the bioleaching process, the jordisite was characterized by X-ray diffractometry (XRD) and scanning electron microscopy (SEM). The results indicate that the addition of Tween 20 has a negative effect on the Fe²⁺-oxidizing activity of A. ferrooxidans, but it is favorable for the sulfur-oxidizing activity of the bacteria. When added in low and high concentrations, Tween 20 enhances and reduces the bioleaching efficiency of jordisite, respectively; thus, it has a significant influence on the bioleaching. After 40 days, a dosage of 30 mg·L^-1 Tween 20 increases the molybdenum extraction yield from 42.21% to 54.10% compared with the bioleaching without Tween 20. It is found that the presence of Tween 20 can accelerate the bio-oxidation of the elemental sulfur generated in the jordisite bioleaching process, consequently increasing the bacteria concentration. Meanwhile, Tween 20 addition reduces the amount of jarosite and elemental sulfur present on the minerals surface, weakening the passivation layer on the minerals surface. This accelerates the jordisite dissolution process, thereby increasing the molybdenum leaching efficiency.