In recent years, triclosan (TCS) has been widely used as an antibacterial agent in personal care products due to the spread of the Coronavirus. TSC is an emerging contaminant, and due to its stability and toxicity, it cannot be completely degraded through traditional wastewater treatment methods. In this study, a novel strain of Enterobacter cloacae was isolated and identified that can grow in high TCS concentrations. Also, we introduced naphthalene dioxygenase as an effective enzyme in TCS biodegradation, and its role during the removal process was investigated along with the laccase enzyme. The change of cell surface hydrophobicity during TCS removal revealed that a glycolipid biosurfactant called rhamnolipid was involved in TCS removal, leading to enhanced biodegradation of TCS. The independent variables, such as initial TCS concentration, pH, removal duration, and temperature, were optimized using the response surface method (RSM). As a result, the maximum TCS removal (97%) was detected at a pH value of 7 and a temperature of 32 °C after 9 days and 12 h of treatment. Gas chromatography-mass spectrometry (GC/MS) analysis showed five intermediate products and a newly proposed pathway for TCS degradation. Finally, the phytotoxicity experiment conducted on Cucumis sativus and Lens culinaris seeds demonstrated an increase in germination power and growth of stems and roots in comparison to untreated water. These results indicate that the final treated water was less toxic.