Rapid and efficient mixing of particles and fluids in a microfluidic system is of great interest for chemical and biochemical analysis. The present paper investigates magnetofluidic mixing induced by a rotating magnetic field from a number of permanent magnets. Numerical simulation shows the complex magnetic field in the mixing chamber. Simulated particle tracing predicts the trajectories of diamagnetic particles in a paramagnetic medium for the different stationary positions of the magnets. The experimentally obtained trajectories show negative magnetophoresis similar to that predicted by the simulation. However, the static configuration of the magnets cannot achieve mixing of the diamagnetic particles. We demonstrated that a rotating magnetic field could yield up to 86% mixing efficiency at a flow rate of 60 μL min−1 using a diluted ferrofluid of only 1% volume concentration.