This paper discusses the vortical flow, mixing and cell culture of Pichia pastoris using a centrifugal microfluidic (CM) chamber. The resultant “spiral toroidal vortex” in the chamber is made up of a primary vortex induced from inertial acceleration/deceleration of the chamber superposed by a secondary toroidal vortex due to Coriolis acceleration acting on the primary vortex. A validated numerical fluid-flow model with minimized numerical diffusion effect has been developed to investigate the flow and consequently mixing of two-color liquids through cyclic constant acceleration-and-deceleration in the same rotation direction until homogeneous mixing of the two liquids in the CM chamber has been established. The specific mixing time is found to improve with increase in acceleration/deceleration and angular span of the chamber. An experimental CM platform with three cell-culture chambers of different angular spans has been built and Pichia pastoris cell culture has been successfully demonstrated. Cell growth can be monitored over time on the extracted samples by measuring the optical density at 600-nm wave-length. Comparing with conventional cell culture, Pichia pastoris cultured on CM platform exhibits a very short lag (cell preparation/budding) phase prior to the log phase (cell growth). While it takes 8 to 12 h for the conventional shake flask in the lag phase, it takes only 2 h for the CM platform irrespective of initial cell concentration (8.1 × 104 to 8.1 × 105/ml), acceleration/deceleration (10 to 32/s2) and angular span of the culture chamber (π/12 to π/4), representing significant time reduction. This is largely attributed to better growth conditions due to enhanced mixing and appropriate shear-stress stimulation from the efficient spiral toroidal vortex.