Thermal pre-flocculation to enable dispersed air flotation is an economical and ecofriendly technology for harvesting microalgae from water. However, the underlying mechanism and optimal conditions for this method remain unclear. In this study, Chlorella vulgaris (C. vulgaris) and Scenedesmus obliquus (S. obliquus) were harvested using a thermal flotation process. The surface structure and characteristics (morphology, electricity, and hydrophobicity) of the microalgae were analyzed using FT-IR (Fourier Transform infrared spectroscopy), SEM (Scanning Electron Microscope), Zeta potential, and a hydrophobic test. Further, Response Surface Methodology (RSM) was used to optimize the flotation process. The hydrophobicity of S. obliquus exceeded that of C. vulgaris; as such, under the thermal pre-flocculation, S. obliquus (88.16%) was harvested more efficiently than C. vulgaris (47.16%). Thermal pre-flocculation denatured the lipids, carbohydrate, and proteins of microalgal cell surfaces. This resulted in a decrease in the electrostatic repulsion between the cells and air bubbles. The highest harvesting efficiency was 91.96% at 70 °C, 1,412 rpm, and 13.36 min. The result of this study demonstrate the potential for economic and ecofriendly harvesting of microalgae for biofuels and other bioproducts industries.