In the microalgae harvesting process, which includes a step for dewatering the algal suspension, directly reusing extracted water in situ would decrease the freshwater footprint of cultivation systems. Among various algae harvesting techniques, membrane-based filtration has shown numerous advantages. This study evaluated the reuse of permeate streams derived from Scenedesmus obliquus (S. obliquus) biomass filtration under bench-scale and pilot-scale conditions. In particular, this study identified a series of challenges and mechanisms that influence the water reuse potential and the robustness of the membrane harvesting system. In a preliminary phase of this investigation, the health status of the initial biomass was found to have important implications for the harvesting performance and quality of the permeate stream to be reused; healthy biomass ensured better dewatering performance (i.e., higher water fluxes) and higher quality of the permeate water streams. A series of bench-scale filtration experiments with different combinations of cross-flow velocity and pressure values were performed to identify the operative conditions that would minimize energy requirements while maximizing water productivity. The optimal conditions, 2.2 m·s⁻¹ and 1.4 bar (1 bar = 10⁵ Pa), respectively, were then applied to drive pilot-scale microfiltration tests to reuse the collected permeate as a new cultivation medium for S. obliquus growth in a pilot-scale photo-bioreactor. The investigation revealed key differences between the behavior of the membrane systems at the two scales (bench and pilot). It indicated the potential for beneficial reuse of the permeate stream as the pilot-scale experiments ensured high harvesting performance and growth rates of biomass in permeate water that were highly similar to those recorded in the ideal cultivation medium. Finally, different nutrient reintegration protocols were investigated, revealing that both macro- and micronutrient levels are critical for the success of the reuse approach.