Abstract Objectives We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. Methods Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3′-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time–kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. Results Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration-dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. Conclusions We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains.