This study investigated waste jasmine flower as a cellulose-based substrate for the reverse osmosis (RO) reject wastewater treatment in a single chamber microbial fuel cell (SCMFC). The two types of MFCs with and without alkaline pretreatment of waste jasmine flower as substrate were operated using a substrate concentration of 200 mg/L (200 mg of substrate in 1 L of RO reject wastewater). A maximum of 68% total dissolved solids (TDS) removal and maximum power density of 2 mW/m² were obtained in the MFC with alkaline pretreated waste jasmine flower as the substrate. The performance of this MFC was found to be better than the MFC without alkaline pretreated waste jasmine flower. Three different concentrations of 200, 250, and 500 mg/L of the selected alkaline pretreated waste jasmine flower substrate were compared for checking the performance of MFC. The most efficient substrate concentration was found to be 200 mg/L. According to the polarization curves, ohmic losses predominated in the SCMFCs for both treated and non-treated substrates.