Marine microalgae from the genus Nannochloropsis are promising candidates for the photoautotrophic production of eicosapentaenoic acid (EPA, C20:5), a polyunsaturated fatty acid known for its numerous health benefits. A recent study demonstrated that Microchloropsis salina can accumulate high amounts of EPA when cultivated in flat-plate gas-lift photobioreactors. This study aimed to characterize an alternative strain, Nannochloropsis oceanica, and compare its biomass and EPA productivity to M. salina. Applying simulated dynamic climate conditions of a repeated sunny summer day in Eastern Australia, N. oceanica was cultivated in LED-illuminated flat-plate gas-lift photobioreactors. The results showed significantly higher biomass growth and EPA contents compared to M. salina. An EPA productivity of 33.0 ± 0.6 mg_EPA L⁻¹ d⁻¹ has been achieved in batch processes with N. oceanica. Scaling up the photoautotrophic process to 8 m² thin-layer cascade photobioreactors resulted in doubled concentrations of N. oceanica biomass compared to laboratory-scale batch processes. This improvement was likely due to the reduced fluid layer depth, which enhanced light availability to the microalgal cells. Using urea instead of nitrate as a nitrogen source further improved the EPA production of N. oceanica in thin-layer cascade photobioreactors, achieving CDW concentrations of up to 17.7 g L⁻¹ and thus a high EPA concentration of 843 mg L⁻¹. These findings highlight N. oceanica as an alternative to M. salina for sustainable EPA production, offering potential for further industrial applications.