This study aims to explore the potential of utilizing rhamnolipid functionalized luffa fibers as an environmentally sustainable solution for removing pharmaceutical contaminants from water. The efficiency of the developed adsorbent for removing acetaminophen has been investigated in batch and fixed bed column modes. The adsorbent has been characterized via FTIR, XRD, and SEM analyses which revealed the rhamnolipid nanomicelles formation with bi-pyramidal morphology and the presence of functional groups on the surface of the adsorbent. The optimum conditions have been reported (pH: 5.0, adsorbent dosage 2 g/L, initial acetaminophen concentration: 20 mg/L, and contact time: 80 min). The highest adsorption capacity for acetaminophen was 37.03 mg/g at the optimized conditions. Fixed-bed column experiments examined flow rate and initial acetaminophen concentration effects, with the Thomas model assessing column performance. This research investigates the potential of RL-LF for effective water remediation, offering insights into practical applications and optimization strategies.