Fault surfaces are rough at all scales, this significantly affecting fault-slip behaviour. However, roughness is only occasionally considered experimentally and then often in experiments imposing a low slip velocity, corresponding to the initiation stage of the earthquake cycle. Here the effect of roughness on earthquake nucleation up to runaway slip is investigated through a series of dry load-stepping biaxial experiments performed on bare rock surfaces with a variety of roughnesses. These laboratory faults reached slip velocities of at least 100 mm/sec. Acoustic emissions were located during deformation on bare rock surfaces in a biaxial apparatus during load-stepping experiments for the first time. Smooth surfaces showed more frequent slip instabilities accompanied by slip bursts and larger stress drops than rough faults. Smooth surfaces reached higher slip velocities and were less inclined to display velocity-strengthening behaviour. The recorded and localized acoustic emissions were characterized by a greater proportion of large-magnitude events, and therefore likely a higher Gutenberg-Richter bGR-value, for smoother samples, while the cumulative seismic moment was similar for all roughnesses. These experiments shed light on how local microscopic heterogeneity associated with surface topography can influence the macroscopic stability of frictional interfaces and the associated microseismicity. They further provide a laboratory demonstration of roughness’ ability to induce stress barriers which can halt rupture, a phenomenon previously shown numerically.