The environmental impacts of algae biofuels have been evaluated by life-cycle assessment (LCA); however, these analyses have overlooked nitrous oxide (N2O), a potent greenhouse gas. A literature analysis was performed to estimate algal N2O emissions and assess the impacts of growth conditions on flux magnitudes. Nitrogen source and dissolved oxygen concentration were identified as possible key contributors; therefore, their individual and combined impacts were evaluated using bench-scale experiments. It was observed that maximum N2O emissions (77.5 μg/g algae/day) occur under anoxic conditions with nitrite. Conversely, minimum emissions (6.25 μg/g algae/day) occur under oxic conditions with nitrate. Aggregated N2O flux estimates were then incorporated into a LCA framework for algae biodiesel. Accounting for “low” N2O emissions mediated no significant increase (<1%) compared to existing GWP estimates; however, “high” N2O emissions mediate an increase of roughly 25%, potentially jeopardizing the anticipated economic and environmental performances of algae biofuels.