Surfactants with their intrinsic ability to solubilize lipid membranes are widely used as antibacterial agents, and their interactions with the bacterial cell envelope are complicated by their differential aggregation tendencies. We present a combined experimental and molecular dynamics investigation to unravel the molecular basis for the superior antimicrobial activity and faster kill kinetics of shorter-chain fatty acid surfactant, laurate, when compared with the longer-chain surfactants studied in contact time assays with live Escherichia coli (E. coli). From all-atom molecular dynamics simulations, translocation events across peptidoglycan were the highest for laurate followed by sodium dodecyl sulfate, myristate, palmitate, oleate, and stearate. The translocation kinetics were positively correlated with the critical micellar concentration, which determined the free monomer surfactant concentration available for translocation across peptidoglycan. Interestingly, aggregates showed a lower propensity to translocate across the peptidoglycan layer and longer translocation times were observed for oleate, thereby revealing an intrinsic sieving property of the bacterial cell wall. Molecular dynamics simulations with surfactant-incorporated bacterial inner membranes revealed the greatest hydrophobic mismatch and membrane thinning in the presence of laurate when compared with the other surfactants. The enhanced antimicrobial efficacy of laurate over oleate was further verified by experiments with giant unilamellar vesicles, and electroporation molecular dynamics simulations revealed greater inner membrane poration tendency in the presence of laurate when compared with the longer-chain surfactants. Our study provides molecular insights into surfactant translocation across peptidoglycan and chain length-induced structural disruption of the inner membrane, which correlate with contact time kill efficacies observed as a function of chain length with E. coli. The insights gained from our study uncover unexplored barrier properties of the bacterial cell envelope to rationalize the development of antimicrobial formulations and therapeutics.