Zero-valent-iron (nZVI) is a candidate antimicrobial agent, and previous works revealed its varying inactivation performance on Gram-negative (G-) and Gram-positive (G+) bacteria, but the underlying mechanism remains ambiguous. Herein, we reported the easier inactivation of Escherichia coli (G-, E. coli) than Staphylococcus aureus (G+, S. aureus) by nZVI, and revealed the key role of cell-nZVI adsorption. nZVI adhered more massively on E. coli surface than on S. aureus, and subsequently led to more pronounced membrane damage of E. coli. Investigations of pH, zeta potential, and ionic strength ruled out the essential contribution of nZVI-bacteria electrostatic interaction due to the different surface charges of E. coli and S. aureus. Three-dimensional excitation emission matrix suggested that the extracellular polymeric substances of E. coli suffered more severe damage by nZVI and lead to greater exposure of membrane. Infrared spectra indicated that nZVI strongly bound with the membrane proteins of E. coli and destroyed the membrane components. By contrast, the bonding between nZVI and S. aureus was minimal because of the dominant multi-layered peptidoglycan. The enhanced nZVI adsorption and membrane disruption would result in magnified reactive oxygen species (ROS) generation and oxidative stress of E. coli. Moreover, the catalase activity normalized by ROS concentration of S. aureus was 14.9-fold higher than that of E. coli after nZVI treatment, suggesting the stronger antioxidative capability of S. aureus. Our findings highlight that the different envelope compositions and antioxidant capacities between G- and G+ bacteria were responsible for their varying susceptibility to nZVI.