Chitosan biguanidine hydrochloride (CGH) has been synthesized by the guanidinylation reaction of chitosan with dicyandiamide. Its synthetic mechanism was discussed. The structures of CGH were characterized by FT-IR and 13 CNMR. In this study, we used citric acid (CA) as a crosslinking agent, mixed with CGH to perform a pad-dry-cure treatment on wool fabric to study its antimicrobial effects with the help of scanning electron microscopy (SEM). The result showed that there was no obvious sign that CGH adhered to the wool fabric if the wool fabrics were not oxidized by hydrogen peroxide. The surface crosslinks of the oxidized wool fibers were relatively coarse, which beneficial for the antimicrobial and antiseptic effects of the wool fabrics.