BackgroundAntimicrobial resistance has become a serious concern and threat to global health. This has triggered the need for the design of new antimicrobials. In this study, poly‐ionic liquid (PIL) homopolymers have been designed by varying the alkyl chain lengths to evaluate their pathogeneses against Gram‐positive bacteria (S. aureus) and Gram‐negative bacteria (E. coli).ResultsThe results demonstrated that the antimicrobial and antibiofilm activities depend on the alkyl chain length of PILs (C10 > C8 > C6). To support the experimental findings, in‐silico analyses have been performed to explore the structural and functional information of bacterial RNA polymerase (RNAP) enzymes. A switch region has been identified between RNAP β subunit (RpoB) and RNAP β’ subunit (RpoC) with 1119 amino acids (aa) and 1504 (2–1505) aa, respectively. The docking analysis reflected the docking energies −5.313 kcal/mol, for RNAP‐poly‐1‐vinyl‐3‐hexyl imidazolium complex, −4.634 kcal/mol for RNAP‐poly‐1‐vinyl‐3‐octyl imidazolium complex and − 3.456 kcal/mol for RNAP‐poly‐1‐vinyl‐3‐decyl imidazolium complex.ConclusionThe synthesized PIL samples exhibited antibacterial and antibiofilm activities against E. coli and S. aureus. However, the effect on E. coli was more prominent in comparison to S. aureus. The results indicated that both electrostatic interaction and hydrophobic effect played a vital role in enhancing the antibacterial as well as antibiofilm activities. In addition, in silico analysis identified the ‘switch region’ between RNAP β subunit (RpoB) and RNAP β’ subunit (RpoC). The novel target appeared to be a binding region for PILs and restricted the synthesis of bacterial RNA. It can be concluded that the imidazolium‐based PILs can be used as safe antibacterial materials in the healthcare sector.This article is protected by copyright. All rights reserved.