Microalgae are a promising sustainable source of biomass while large-scale harvesting of microalgal biomass is a major technological and economic challenge. In this study, a flocculation method and mechanism for the formation of compact mega flocs was developed using two oppositely charged polyelectrolytes, quaternary ammonium chitosan (N-[(2-hydroxy-3-trimethyl ammonium) propyl] chitosan, HTCC) and xanthan gum (XG). With 8 mg/L HTCC and 16 mg/L XG, Scenedesmus cells could be flocculated into mega flocs with diameters larger than 5 mm and were harvested by 500-mu m-pore-sized sieves with an efficiency over 95%. The flocs were generated within 12 s and resisted strong hydraulic shear (stirring at 960 rpm), reducing energy consumption and handling time for the subsequent separation. With HTCC or XG alone, the harvest efficiency was consistently lower than 30%. The flocculation mechanism was investigated by changing the order in which the HTCC, XG, and Scenedesmus cells were added, and by fluorescence staining, scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The results showed that the mega flocs were formed by the crosslinking of HTCC and XG, based on their long-chain molecular structure and electrostatic interaction. Scenedesmus cells were enmeshed in the polymer matrix of HTCC and XG and then aggregated into large flocs. Thus, an effective, eco-friendly, and sustainable flocculation strategy for microalgae biomass was developed using the biodegradable components HTCC and XG.