β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure-activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N2-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N2-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01-0.05 μmol/mL. Our work highlights that N1-N1 dimerization and N2-benzylation significantly enhanced the antimicrobial effects of compounds.