AbstractA series of novel 2‐alkyl‐5‐((phenylsulfonyl)oxy)‐1H‐indole‐3‐carboxylate derivatives were synthesized and evaluated for their cytotoxicity, antibacterial and anti‐biofilm activities. Some of these compounds were able to inhibit biofilm formation of the tested strains with biofilm inhibitory concentration in the range of 12.5–50 μM. The results indicated N‐methylpiperazine moiety was the key factor affecting the compound‘s activity. Compounds 5 l and 5 m with F substituent at the phenyl ring at 5‐position of indole ring, presented the most potent biofilm inhibitory activity (more than 90 % inhibition percentage) against E. faecalis 16C51 at biofilm inhibitory concentration (12.5 μM). Furthermore, compounds 5 f and 5 h with Br/CH3 substituent at the phenyl ring at 1‐position of indole ring, showed the greatest biofilm inhibitory activity (more than 85 % inhibition percentage) against MRSA YUSA145 at biofilm inhibitory concentration (12.5 μM).