Schizochytrium has arisen as a valuable alternative to fish as a source of polyunsaturated fatty acids. In the present study, Schizochytrium limacinum CO3-H biomass was processed by direct transesterification affording a transesterified algal oil, from which docosahexaenoic acid (DHA) and docosapentaenoic acid n-6 (DPA n-6) ethyl esters were isolated using high performance countercurrent chromatography (HPCCC). A multiple sequential injection HPCCC separation method was developed combining two elution modes (reverse phase and extrusion). During the reverse phase elution, the lower phase of a biphasic solvent system (n-heptane:acetonitrile, ratio 5:5, v/v) was used as the mobile phase. During the initial reverse phase elution, the two target compounds were obtained, which was followed by the extrusion of the stationary phase by switching the pumping of the mobile phase to the stationary phase, while maintaining the column rotation. Once the column was refilled with stationary phase, a new hydrodynamic equilibrium condition was again reached for a new separation cycle. Ten consecutive sample injections (1000 mg of algal oil, each) were performed in this way leading to the separation of DHA ethyl ester (1, 797 mg, 99% purity) and DPA n-6 ethyl ester (2, 164 mg, 97% purity) with recoveries of 99% and 92%; respectively. The process throughput of algal oil was 1.149 g/h while the efficiency per gram of two target compounds was 0.110 g/h. Environmental risk and process evaluation factors were used for evaluation of the separation process. Overall, this separation strategy may represent a useful model for the co-production of DHA and DPA n-6 from microalgae oil under a biorefinery perspective because the potential co-production of other valuable compounds present in both the algal oil and biomass may contribute to enhancement of the economics of the approach.