To assess the fate and behavior of engineered nanoparticles in the environment, it is important to examine the physicochemical and toxicological transformation of nanoparticles as they age in seawater. In this study, we investigated how aging and seawater conditions altered the physiochemical structure of nanoparticles and affected their interactions with bacteria. For this purpose, zinc oxide nanoparticles were aged under different seawater conditions by keeping them in 1%, 10%, and 100% seawater for 1 day and 20 days. The main physicochemical parameters (surface chemistry, chemical composition, particle size, and zeta potential) and toxicity of aged nanoparticles towards gram-negative Pseudomonas aeruginosa and gram-positive Staphylococcus aureus were examined. The results indicated that aged zinc oxide nanoparticles in various concentrations of seawater changed their surface chemistry, chemical composition, particle size, and zeta potentials. Growth inhibition results were observed in that the inhibition of gram-negative (Pseudomonas aeruginosa) bacteria was higher compared with the gram-positive (Staphylococcus aureus) bacteria, and Staphylococcus aureus activated with the aged zinc oxide nanoparticles. Also, the results showed that the key biochemical factors affected by the aging and seawater concentration.