In this study, polycaprolactone (PCL) microfibrous scaffolds with berberine were fabricated to mimic the natural extracellular matrix (ECM) architecture and provide antimicrobial activity for annulus fibrosus tissue engineering. Morphological characterization showed that there was a significant decrease of the average fiber diameter in the berberine-loaded microfibrous scaffolds (B-MFS, 0.40 +/- A 0.02 mu m) compared with that of the non-drug-loaded microfibrous scaffolds (MFS, 1.89 +/- A 0.15 mu m). The antimicrobial activity, drug release profile, and biocompatibility of the scaffolds were evaluated. The B-MFS displayed excellent antimicrobial activities against Gram-positive bacteria (S. aureus 6538), Gram-negative bacteria (E. coli 15597), fungus (C. albicans 10231) and drug-resistant bacteria (methicillin-resistant S. aureus BAA-811, or MRSA BAA-811). After seeding with porcine AF cells, the in vitro biocompatibility of the scaffolds was determined by measuring cell attachment, cell proliferation, and ECM production. Total cell number, sGAG and collagen content gradually increased from day 1 to day 7 in both groups. When compared to MFS, the B-MFS group displayed higher levels of cell proliferation throughout the experimental period. These results indicate that PCL microfibrous loaded with berberine are novel biocompatible scaffolds with a broad-spectrum antimicrobial activity for AF tissue engineering.