Bioleaching has been applied as a successful technique for metal recovery from various mineral sources like low-grade ores, waste materials and tailings. Mixed cultures of bioleaching microorganisms have a high performance in mineral dissolution. Thus far, microbial interactions in bioleaching communities are poorly understood. In this paper, the acidophilic archaeon Ferroplasma acidiphilum and the bacteria Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans were chosen to study their interactions during pyrite leaching. The initial attachment to pyrite and pyrite leaching efficiency of pure and mixed populations were investigated. The data indicate: (i) attachment and bioleaching efficiency of L. ferriphilum was reduced in the presence of F. acidiphilum. However, the combination of F. acidiphilum and S. thermosulfidooxidans showed increased leaching, although the initial attachment rate was reduced, when compared to pure cultures. Thus, synergistic or antagonistic interactions may exist between F. acidiphilum and S. thermosulfidooxidans or F. acidiphilum and L. ferriphilum, respectively; (ii) pre-established biofilms of L. ferriphilum inhibited initial attachment to pyrite by cells of F. acidiphilum and did not promote pyrite leaching by F. acidiphilum. In contrast, inactivated biofilm cells of S. thermosulfidooxidans enhanced pyrite bioleaching by F. acidiphilum; (iii) adhesion forces of cells to an AFM tip (Si3N4) seemed to be not correlated to attachment and bioleaching capacity; and (iv) lectins were applied to show and distinguish single species in mixed biofilm populations. Physical contact between cells of S. thermosulfidooxidans and F. acidiphilum was visible.