Magnetic separation of microalgae using magnetite is a promising harvesting method as it is fast, reliable, low cost, energy-efficient, and environmentally friendly. In the present work, magnetic harvesting of three green algae ( and ) and one cyanobacteria () has been studied. The biomass was flushed with clean air using a 0.22 μm filter and fed CO for accelerated growth and faster reach of the exponential growth phase. The microalgae were harvested with magnetite nanoparticles. The nanoparticles were prepared by controlled co-precipitation of Fe and Fe cations in ammonia at room temperature. Subsequently, the prepared FeO nanoparticles were coated with polyethyleneimine (PEI). The prepared materials were characterized by high-resolution transmission electron microscopy, X-ray diffraction, magnetometry, and zeta potential measurements. The prepared nanomaterials were used for magnetic harvesting of microalgae. The highest harvesting efficiencies were found for PEI-coated FeO. The efficiency was pH-dependent. Higher harvesting efficiencies, up to 99%, were obtained in acidic solutions. The results show that magnetic harvesting can be significantly enhanced by PEI coating, as it increases the positive electrical charge of the nanoparticles. Most importantly, the flocculants can be prepared at room temperature, thereby reducing the production costs.