In this study aimed to the synthesis of novel bis‐nicotinonitrile derivatives starting from 1,3‐bis‐chalcones and investigate their antimicrobial activity potentials. Bis‐nicotinonitrile derivatives were synthesized from 1,3‐bis‐chalcone derivatives reacted with malononitrile in ethanol in the presence of potassium carbonate. Characterization of the obtained compounds was carried out by spectroscopic methods. In total, 12 new 1,3‐phenylene‐bis‐nicotinonitrile derivatives were synthesized. Finally, the antimicrobial and antibiofilm activities of synthesized 1,3‐phenylene‐bis‐nicotinonitrile derivatives were tested against 5 microorganisms. As a result, it was observed that the synthesized new nicotinonitrile derivatives were quite active especially in terms of antibiofilm activity.