The dynamic nature of bacterial lipid membranes significantly impacts the efficacy of antimicrobial therapies. However, traditional assay methods often fall short in replicating the complexity of these membranes, necessitating innovative approaches. Herein, we successfully fabricated model bacterially supported lipid bilayers (SLBs) that closely mimic the characteristics of Gram-positive bacteria using the solvent-assisted lipid bilayer (SALB) technique. By employing a quartz crystal microbalance with dissipation and fluorescence microscopy, we investigated the interactions between these bacterial mimetic membranes and long-chain unsaturated fatty acids. Specifically, linolenic acid (LNA) and linoleic acid (LLA) demonstrated interaction behaviors correlated with the critical micelle concentration (CMC) on Gram-positive membranes, resulting in membrane remodeling and removal at concentrations above their respective CMC values. In contrast, oleic acid (OA), while showing similar membrane remodeling patterns to LNA and LLA, exhibited membrane insertion and CMC-independent activity on the Gram-positive membranes. Particularly, LNA and LLA demonstrated bactericidal effects and promoted membrane permeability and ATP leakage in the bacterial membranes. OA, characterized by a CMC-independent activity profile, exhibited potent bactericidal effects due to its robust penetration into the SLBs, also enhancing membrane permeability and ATP leakage. These findings shed light on the intricate molecular mechanisms governing the interactions between long-chain unsaturated fatty acids and bacterial membranes. Importantly, this study underscores the potential of using biologically relevant model bacterial membrane systems to develop innovative strategies for combating bacterial infections and designing effective therapeutic agents.