The realization of efficient mixing of samples inside a microfluidic channel is essential for performing numerous biological assays in miniaturized total analysis systems. The low Reynolds number flows at the microscale create laminar streams inside the microchannel, limiting flow mixing to a molecular diffusion level. In this paper, we propose a simple and efficient acoustofluidic mixing technique inside a single-layered polydimethylsiloxane (PDMS) microfluidic channel. The proposed surface acoustic wave (SAW)-based system composed of a straight interdigitated transducer (IDT) is positioned beneath the PDMS microchannel. Fluorescein dye dissolved in deionized water (sample fluid) and deionized water (sheath fluid) was introduced through the first and second inlets of the PDMS microchannel, respectively. Their flow rates were controlled such that the sample fluid with fluorescein dye was hydrodynamically focused close to the bottom of the microchannel by the sheath fluid. High-frequency (140 MHz) SAWs, generated from the IDT placed right beneath the first outlet, mixed the two fluids under the influence of strong acoustic streaming flows. The mixed samples were then collected at the two outlet ports for further analysis of the mixing efficiency. The developed acoustofluidic mixing device required an input voltage of 12 Vpp at a total flow rate of 50 μl/min to realize complete mixing. At a similar applied voltage, the throughput of the proposed device could be further increased to 200 μl/min with a mixing efficiency of >90%.