A series of N-substituted N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidine derivatives bearing sulfonamide moiety have been screened in vitro for antibacterial activity against isolates from patients with infections of oral cavity, respiratory tract and intestinal tract. The majority of compounds exhibited good antibacterial potency. 1-[4-Chloro-5-methyl-2-(4-trifluoromethylbenzylthio)benzenesulfonyl]-3-(3-sulfamoylphenyl)guanidine (13) showed very strong activity, with MIC ≤ 6.2 μg/ml against eleven bacteria strains belonged to Gram-positive anaerobes and aerobes. Furthermore, compound 13 exhibited promising activity toward highly resistant microorganisms such as methicillin-resistant Staphylococcus aureus and Enterococcusfaecalis. It was found that Parvimonas micra, Finegoldia magna, Peptostreptococcus anaerobius, Propionibacterium acnes showed the highest susceptibility toward the investigated guanidines.