The mobility of tetracycline antibiotics (TCs) in saturated aquifers is possibly affected by the presence of biosurfactants, which are widespread in the aquatic/soil environments. This study investigated the mobility characteristics of various tetracyclines—specifically tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC)—within quartz sand columns in the presence of rhamnolipid, a common biosurfactant. Exogenous rhamnolipid significantly inhibited the transport of the three TCs over the pH range of 5.0–9.0 (e.g., the mass of retained TC, OTC, and CTC increased from 32.6 %, 26.9 %, and 39.2 % (in the absence rhamnolipid) to 39.4 %, 38.9 %, and 51.7 % (in the presence of rhamnolipid), respectively). This observation could be attributed to the bridging effects of this biosurfactant. Specifically, the hydrophilic head of rhamnolipid molecules is likely associated with the surfaces of sand grains through surface complexation and/or hydrogen bonding interactions. Accordingly, the hydrophobic moieties of the deposited rhamnolipid molecules (i.e., the aliphatic chains) interact with the hydrophobic groups of TCs molecules via hydrophobic interactions. Interestingly, the extent of the inhibitory effect on CTC mobility was greater than that on OTC and TC, which was related to the different hydrophobic characteristics of the three antibiotics. Furthermore, the inhibitory effect of rhamnolipid on the transport of TCs diminished as the pH of the background solution increased. This observation was attributed to the weakened bridging effects, resulting from the reduced deposition of the biosurfactant on the sand surfaces. Additionally, the cation-bridging mechanism involved in the retention of TCs in the addition of rhamnolipid when the background electrolyte was Ca2+ (i.e., Ca2+ ions served as bridging agents between the deposited rhamnolipid molecules and TCs). The insightful findings enhance our understanding of the critical roles of biosurfactants in influencing the environmental dynamics and ultimate fate of conventional antibiotic pollutants within groundwater systems.