AbstractInhibitory potential of 19 benzimidazoles against bovine pancreatic deoxyribonuclease I (DNase I) was investigated in vitro. Three compounds inhibited DNase I with IC50 below 100 μM and proved to be more potent DNase I inhibitors than crystal violet (IC50 = 351.82 ± 29.41 μM), used as a positive control. Compound 9 showed the most potent DNase I inhibition with an IC 50 value of 79.46 ± 11.75 μM. To further explore the relationship between inhibitory activity and 2D pharmacophore features, Pharma/E‐State R‐group quantitative structure‐activity relationship (RQSAR) models were generated and validated using Schrödinger Suite. RQSAR models showed a significant enhancement of benzimidazoles activity using hydrogen‐bond acceptor substituents at the R2, R3, and R4 positions, or aryl substituents at the R4 position. The Site Finder module and molecular docking defined the benzimidazoles interactions with the most important catalytic residues of DNase I, including H‐acceptor interaction with residue His 134 and His 252 and/or H‐donor interaction with residue Glu 39. We also found a positive correlation between IC50 inhibition values and relative binding free energies of the most active benzimidazoles. In addition, a molecular dynamics simulation was performed for DNase I‐compound 9 docking complex in Desmond. Trajectory analysis showed that docking complex and intermolecular interactions were stable throughout the entire production part of simulations. Furthermore, the results of protein structure alignment module suggested the potential translational impact of benzimidazoles against human DNase I. Due to the significant involvement of DNase I in the pathophysiology of many disease conditions, benzimidazoles as DNase I inhibitors could have potential therapeutic applications.