Ultrafast particle and cell concentration is essential to the success of subsequent analytical procedures and the development of miniaturized biological and chemical sensors. Here, surface acoustic wave (SAW) devices were used to excite a MHz-order acoustic wave that propagates into a microlitre droplet to drive spatial concentration and separation of two different sized suspended microparticles. The rapid concentration process, occurring within just three seconds to facilitate spatial partitioning between the two particle species, exploited two acoustic phenomena acting on the suspended particles: the drag force arising from acoustic streaming and the acoustic radiation force, both driving particles in different directions. This study elucidates the very intricate and interesting interplay of physics between fluid drag and acoustic forcing on the particles within a droplet, and, for the first time, demonstrates the existence of a frequency-dependent crossover particle size that can be used to effect species partitioning: depending on the operating frequency of the SAW device and the particle size, it is possible to cause one phenomenon to dominate over the other. A theoretical analysis revealed the extent to which each force would affect the particle trajectory (particle size range: 2-31 μm), subsequently verified through experimentation. Based on these findings, 6 and 31 μm polystyrene particles were successfully partitioned in a water droplet using a 20 MHz SAW device. This study reveals the suitability of using acoustic actuation methods for the useful partitioning of particle species within a discrete fluid volume.