A novel derivative of chitosan (CTS), ortho-biguanidinyl benzoyl chitosan hydrochloride (o-BGBCH), was prepared with CTS and ortho-biguanidinyl benzoyl chloride, which was synthesized by acid chloride reaction of ortho-biguanidinyl benzonic acid hydrochloride (o-BGBA), as starting material in the medium consisted of methylsulfonic acid (MeSO3H) and dimethyl sulfoxide. Structure of o-BGBCH was characterized by FT-IR and H-1 NMR, molecular weight of o-BGBCH was determined through gel permeation chromatography, and degree of substitution (DS) of guanidinylation of o-BGBCH was measured using elemental analysis technique. Meanwhile, the minimum inhibitory concentration of o-BGBCHs against Escherichia coli, a Gram-negative bacterium, and Staphylococcus aureus, a Gram-positive bacterium, were ascertained by agar plate method. Compared with CTS hydrochloride, o-BGBCHs had much stronger antimicrobial activities, and these activities improved with the increase of their DS of guanidinylation. When the DS of o-BGBCH reached or exceeded 32.7 %, its antibacterial activities against the tested bacteria were higher than that of Bromo-Geramium.