A simple and facile protocol for the regioselective synthesis of diastereomeric 3-substituted indole derivatives is described by the three-component condensation of 3-(cyanoacetyl)indole, aldehydes, and kojic acid in the presence of ammonium acetate as catalyst. The synthesized compounds were tested for their antibacterial effects against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. One compound showed good activity against B. subtilis compared with the standard drugs gentamicin and chloramphenicol.