This study represents synthesis, characterization, screening of antibiofilm efficacy, and cytotoxicity of iridium bis-terpyridine complexes. The complexes were characterized by NMR, MS, FTIR, UV/Visible, and fluorescence spectroscopies. The efficacy of biofilm inhibition and eradication of iridium complexes was evaluated using a crystal violet assay test and verified by fluorescence microscopy. Cytotoxicity and apoptosis analysis of iridium complexes were determined in this study. The results of our study revealed that three iridium complexes had the potential to inhibit biofilm formation and moderate the ability to destroy pre-formed biofilm of S. aureus ATCC 29,213. 250 µM concentration of synthesized complexes showed the highest antibiofilm activity (75% for Ir1, 90% for Ir2, and 71% for Ir3). The significant inhibition obtained at 6.25 µM concentration of Ir2 and Ir3 revealed the potential of our samples. Also, Ir1 and Ir2 complexes had a good capacity to destroy pre-formed biofilm. The results clearly showed that iridium complexes have cytotoxic activity towards colon cancer (Caco-2) and liver cancer (HepG2) cell lines without affecting non-cancerous cells (HEK293) at applied doses. Moreover, tested compounds induced apoptosis in these cancer cells. All of these results showed that iridium complexes had possessed the ability to inhibit or destroy pre-formed biofilm and could be developed as an effective agent against bacterial biofilms. Moreover, these pure substances may have valuable anti-cancer activity and it should be confirmed with further studies for therapeutic effects. © 2021, The Author(s), under exclusive licence to Springer Nature B.V.