A conducting polymer-carbon nanofiber-based nanocomposite was developed as an efficient microbial fuel cell (MFC) anode for the production of bioenergy using a microbial catalyst. The activated carbon nanofiber (ACNF), with and without carbon nanotubes (CNTs) embedded in them were prepared by stabilizing and activating the non-woven web of the polyacrylonitrile (PAN)-based carbon nanofibers prepared by electrospinning. The polypyrrole (PPy)-coated ACNF/CNT (PPy-ACNF/CNT) nanocomposites were synthesized by an in-situ chemical polymerization, and the electrochemical properties and performance of the modified anode when used in an MFC system were then evaluated. The power density of the PPy-ACNF/CNT nanocomposite anode showed a 1.8 fold increase as compared to that of the commercial graphite felt. The high electrode conductivity, stability, and biocompatibility of the modified anodes make them attractive for the practical MFC applications.