Microbial Fuel Cells (MFCs) are biological fuel cells based on the oxidation of fuels by electrogenic bacteria to generate an electric current in electrochemical cells. There are several methods that can be employed to improve their performance. In this study, the effects of gold surface modification with different thiol molecules were investigated for their implementation as anode electrodes in micro-scale MFCs (mu MFCs). Several double-chamber mu MFCs with 10.4 mu L anode and cathode chambers were fabricated using silicon-microelectromechanical systems (MEMS) fabrication technology. mu MFC systems assembled with modified gold anodes were operated under anaerobic conditions with the continuous feeding of anolyte and catholyte to compare the effect of different thiol molecules on the biofilm formation ofShewanella oneidensisMR-1. Performances were evaluated using polarization curves, Electrochemical Impedance Spectroscopy (EIS), and Scanning Electron Microcopy (SEM). The results showed that mu MFCs modified with thiol self-assembled monolayers (SAMs) (cysteamine and 11-MUA) resulted in more than a 50% reduction in start-up times due to better bacterial attachment on the anode surface. Both 11-MUA and cysteamine modifications resulted in dense biofilms, as observed in SEM images. The power output was found to be similar in cysteamine-modified and bare gold mu MFCs. The power and current densities obtained in this study were comparable to those reported in similar studies in the literature.