The use of zero-valent iron nanoparticles (nZVI) is an emerging application for environmental remediation. In the present work, the effect of mild nZVI conditions (2.5, 10, and 25 mg L−1) on the bacteria biofilm and growth of both planktonic and sessile cells based on a Pseudomonas putida (P. putida) model was studied. Core shell nZVI nanoparticles (25 nm in size) were purchased and characterized. P. putida biofilm was growth in Luria–Bertani (LB) in absence and presence of nZVI at different concentrations, 2.5, 10, and 25 mg L−1 for 7 days. The secretion of exopolysaccharides (EPS), the population of planktonic cells and sessile cells, and biofilm structure were investigated. nZVI did not inhibit planktonic cells growth at all nZVI concentrations, suggesting nZVI as an inert against planktonic cells. More importantly, the EPS production and population of sessile cells in nZVI-biofilm were considerably higher than those in control biofilm. A dense and confluent biofilm structure for nZVI-biofilm was developed even at 2.5 mg L−1 of nZVI concentration, probably to prevent the penetration of nZVI into the biofilm matrix. The present work has revealed that nZVI plays a role in a promotion of biofilm production. Furthermore, a decrease in population of P. putida in nZVI-biofilm was delayed when compared to that in control biofilm. The 1-log reduction was reached at 30 and 60 days for control biofilm and nZVI-biofilm, respectively. In conclusion, nZVI provided a positive action for dense biofilm formation and consequently enhancement of bacteria cell survival.