A novel anionic gemini surfactant containing an ester bond in the spacer group was synthesized using cardanol as the raw material and characterized by IR, 1H NMR and 13C NMR. The surface properties of the gemini surfactant were investigated and compared with its corresponding single chain surfactant counterpart. It was found that this novel gemini surfactant exhibited a low critical micelle concentration value (1.9 mM) and good efficiency in reducing surface tension of water (33.6 mN/m). The gemini surfactant was found to have antimicrobial activity against Gram‐negative (Escherichia coli and Pseudomonas aeruginosa), Gram‐positive (Bacillus subtilis and Staphylococcus aureus) bacteria and fungi (Aspergillus niger, Aspergillus flavus, Candida albicans and Rhizopus stolonifer). The gemini as well as the corresponding single chain surfactant showed good antimicrobial activity against all pathogenic microorganisms studied and can be employed as an antimicrobial agent. The synthesized novel anionic gemini surfactant possesses an excellent wettability and low foamability.