Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid of nutritional and physiological relevance for humans. It is commercially produced mainly from fish oil, but new sustainable sources are required. In this study, pure EPA ethyl ester was produced from Nanofrustulum shiloi biomass by integrating two separation steps via high performance countercurrent chromatography (HPCCC). The HPCCC first step consisted of a 10 separation cycles (1200 mg sample per cycle) process, each cycle consisting of three stages comprising EPA fraction separation in reversed phase elution mode (lower polar phase used as mobile phase); with stationary phase extrusion (by switching the pumping of the mobile phase to the stationary phase, while maintaining the column rotation); and hydrodynamic equilibrium achievement inside the column. The process led to 982.1 mg of a fraction rich in EPA with a purity of 71%. To improve the purity of the EPA fraction, an HPCCC second step was performed leading to 698.97 mg of EPA ethyl ester with a purity of 99%, and a recovery of 98%. To avoid unnecessary waste of solvents, liquid phases were formulated based on a priori NMR measurement. The HPCCC process throughput was 0.936 g h–1, while the efficiency per gram of EPA was 0.054 g h–1. Environmental risk and process evaluation factors were used for the evaluation of the separation process. Overall, this isolation approach may represent a useful model for the efficient production of pure EPA from microalgae.