Microalgae are excellent sources of polyunsaturated fatty acids (PUFAs), but only a few species have been thoroughly investigated in controlled photobioreactor conditions. In this work, the cold-adapted microalga Koliella antarctica (Trebouxiophyceae) was cultivated at 15 °C to optimize growth and PUFA production in bubble-tube and flat-plate photobioreactors. The impact of nitrogen starvation, phosphorus starvation, salinity, and light intensity on the growth, fatty acid, and protein content was investigated. After culture optimization, a maximum biomass productivity of 2.37 g L−1 day−1 and maximum cell density of 11.68 g L−1 were achieved. Among all conditions tested, the maximum total fatty acid (TFA) content measured 271.9 mg g−1 dry weight in the late stationary phase. Nitrogen and phosphorus starvation strongly induced neutral lipid (TAG) accumulation, up to 90.3% of TFA, which mostly consisted of the monounsaturated fatty acid C18:1n−9 (oleic acid, OA). PUFAs were also abundant and together accounted for 30.3–45.8% of total triacylglycerol (TAG). The highest eicosapentaenoic acid (EPA) content (C20:5n−3) amounted to 6.7 mg g−1 dry weight (4.9% TFA) in control treatments, while the highest arachidonic acid (ARA) content (C20:4n−6) was 9.6 mg g−1 dry weight (3.5% TFA) in the late stationary phase. Phosphorus starvation was an effective strategy to obtain high total fatty acid yields (mg L−1) while maintaining the protein, total PUFA, and omega-3 fatty acid contents.