A series of 1,3-bis-chalcone derivatives (3a-i, 6a-i and 8) were synthesized and evaluated antimicrobial, antibiofilm and carbonic anhydrase inhibition activities. In this evaluation, 6f was found to be the most active compound showing the same effect as the positive control against Bacillus subtilis and Streptococcus pyogenes in terms of antimicrobial activity. Biofilm structures formed by microorganisms were damaged by compounds at the minimum inhibitory concentration value between 0.5% and 97%.1,3-bis-chalcones ( 3a-i, 6a-i and 8) showed good inhibitory action against human (h) carbonic anhydrase (CA) isoforms I and II. hCA I and II were effectively inhibited by these compounds, with K-i values in the range of 94.33 +/- 13.26 to 787.38 +/- 82.64nM for hCA I, and of 100.37 +/- 11.41 to 801.76 +/- 91.11nM for hCA II, respectively. In contrast, acetazolamide clinically used as CA inhibitor showed K-i value of 1054.38 +/- 207.33nM against hCA I, and 983.78 +/- 251.08nM against hCA II, respectively.