Acid bioleaching of a low-grade and polymetallic sulfide concentrate was studied, in order to determine suitable feed material particle size and pH for efficient leaching of valuable metals. The sulfide concentrate consisted of pyrite (50 wt %), pyrrhotite (31 wt %), quartz (10 wt %) and lower amounts of cobalt, nickel, zinc and copper (each <1 wt %). After adaptation of microorganisms in shake flasks, stirred tank tests were conducted at different pH levels and supplementing feed material at different particle sizes (milled to d80 < 150 µm, <50 µm, <28 µm, <19 µm). The operation at pH 1.8 was seen prone to iron precipitation, while this was not observed at a pH between 1.3 and 1.5. Additional milling to decrease particle size from the initial d80 < 150 µm had a major positive effect on cobalt- and nickel-leaching yields, proposing that at least d80 < 28 µm should be targeted. The best leaching yields for the main economic elements, cobalt and nickel, were 98% and 94%, respectively, reached with d80 < 19 µm at pH 1.3. However, it was considered that at pH 1.5, similar results could be obtained. This research sets the basis for continuing the experiments at a continuous pilot scale.