Energy-storage metabolites such as neutral lipids and carbohydrates are valuable compounds for liquid biofuel production. The aim of this work is to elucidate the main biological responses of two algae species known for their effective energy-rich compound accumulation in nitrogen limitation and day−night cycles: Nannochloropsis gaditana, a seawater species, and Parachlorella kessleri, a freshwater species. Lipid and carbohydrate production are investigated, as well as cell resistance to mechanical disruption for energy-rich compound release. Nitrogen-depleted N. gaditana showed only a low consumption of energy-storage molecules with a non-significant preference for neutral lipids (TAG) and carbohydrates in day−night cycles. However, it did accumulate significantly fewer carbohydrates than P. kessleri. Following this, the highest levels of productivity for N. gaditana in chemostat cultures at four levels of nitrogen limitation were found to be 3.4 and 2.2 × 10−3 kg/m2·d for carbohydrates and TAG, respectively, at 56%NO3 limitation. The cell disruption rate of N. gaditana decreased along with nitrogen limitation, from 75% (at 200%NO3) to 17% (at 13%NO3). In the context of potentially recoverable energy for biofuels, P. kessleri showed good potential for biodiesel and high potential for bioethanol; by contrast, N. gaditana was found to be more efficient for biodiesel production only.