Bioclogging can significantly reduce the permeability of porous media, restricting the application of artificial recharge. In this study, the quantitative efficiency and mechanism by which rhamnolipid (RL) biosurfactant prevents bioclogging during artificial recharge were investigated in both batch-reactor and column systems. The batch experiments indicated that the adsorption of bacteria onto quartz sand surface was significantly reduced in the presence of RL. The adsorption isotherm data were well fitted to the Freundlich model. The Freundlich sorption affinities (Kf) of bacteria in the presence of 0, 20, 40, and 80 mg/L RL were 3.20, 1.55, 0.79, and 1.21, respectively. In the microplate biofilm experiments, the addition of 40 mg/L of RL (40 RL) reduced the total biomass to 17.4%. The percolation sand column experiments demonstrated the efficacy of RL alleviating bioclogging. More time was taken (18 h) for the 40 RL group to achieve a relative hydraulic conductivity (K′) of 0.20 compared with the group without the addition of RL (0 RL) (12 h). Meanwhile, the accumulation of bacterial cells and the production of extracellular polymeric substances (EPS), which are the main contributors to bioclogging, were lower in groups with RL than in those without RL. The optimal concentration of RL is 40 mg/L. The decreasing degree of K′ in different experimental groups was linearly correlated with RL-dependent cell surface hydrophobicity (CSH). The results of both batch and column experiments indicate that the RL reduced the adsorption capacity of bacteria onto the surface of porous media by modifying the hydrophobicity of the bacteria, reducing bioclogging in artificial recharging. This study therefore provides insights and guidances for reducing bioclogging, thereby enhancing the efficiency of artificial recharge.