Seventeen benzofuran derivatives were synthesized and screened for their antibacterial activities against Escherichia coli, Staphylococcus aureus, Methicillin-resistant S. aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Seven of them have showed excellent antibacterial activities compared to the positive controls (Cefotaxime and Sodium Penicillin). The substitutions at C-6 and C-3 positions of these derivatives were found to greatly impact on the antibacterial activity and strains specificity, respectively. Specifically, compounds bearing a hydroxyl group at C-6 (5a, 5b, 5c and 12) offered excellent antibacterial activities against all five above-mentioned strains (MIC(80) = 0.78-12.5 ug/mL), and those with imine (15) and (3, 4, 5-trimethoxyphenyl) methanone (7e), respectively, at C-3 position showed selective activity against S. aureus among five tested strains with great MIC(80) values (3.12-12.5 ug/mL).