Aims To determine the antimicrobial potency of a surface‐anchored quaternary ammonium salt (SAQAS)‐based biocide during in vitro wet and dry fomite assays and to determine the mechanism of killing bacteria on the surface. Methods and Results Wet and dry fomite assays were established in vitro for a commercially available biocide (SAQAS‐A) applied to glass and low‐density polyethylene (LDPE) surfaces. Both wet and dry fomite tests showed the active killing of Gram‐positive and Gram‐negative bacteria but not endospores. Assays measuring membrane permeability (ATP and DNA release), bacterial membrane potential and bacterial ROS production were correlated with the time‐to‐kill profiles to show SAQAS‐A activity in suspension and applied to a surface. Conclusions SAQAS‐A is an effective biocide against model strains of vegetative bacteria. The killing mechanism for SAQAS‐A observed minimal membrane depolarization, a surge in ROS production and assessment of membrane permeability supported the puncture of cells in both suspension and surface attachment, leading to cell death. Significance and Impact of the study SAQAS represents effective surface biocides against single challenges with bacteria through a mechanical killing ability that supports real‐world application if their durability can be demonstrated to maintain residual activity.