Biofuels from microalgae are potentially important sources of liquid renewable energy. Algae are not yet produced on a large scale, but research shows promising results. This study assesses the blue water footprint (WF) and land use of algae-based biofuels. It combines the WF concept with an energy balance approach to determine the blue WF of net energy. The study considers open ponds and closed photobioreactors (PBRs). All systems have a positive energy balance, with output-input ratios ranging between 1.13 and 1.98. This study shows that the WF of algae-based biofuels lies between 8 and 193 m3/GJ net energy provided. The land use of microalgal biofuels ranges from 20 to 200 m2/GJ net energy. For a scenario in which algae-based biofuels provide 3.5% of the transportation fuels in the European Union in 2030, the system with the highest land productivity needs 17,000 km2 to produce the 850 PJ/yr. Producing all algae-based biofuels through the system with the highest water productivity would lead to a blue WF of 7 Gm3/yr, which is equivalent to 15% of the present blue WF in the EU28. A transition to algae-based transportation fuels will substantially increase competition over water and land resources.