Silver nanoparticles (Ag-NPs) can be considered as a cost-effective alternative to antibiotics. In the presence of Fe(III)-citrate and Ag+,Klebsiella oxytocaDSM 29614 produces biogenic Ag-NPs embedded in its peculiar exopolysaccharide (EPS).K. oxytocaDSM 29614 was cultivated in a defined growth medium-containing citrate (as sole carbon source) and supplemented with Ag(+)and either low or high Fe(III) concentration. As inferred from elemental analysis, transmission and scanning electron microscopy, Fourier transform infrared spectrometry and dynamic light scattering, Ag-EPS NPs were produced in both conditions and contained also Fe. The production yield of high-Fe/Ag-EPS NPs was 12 times higher than the production yield of low-Fe/Ag-EPS NPs, confirming the stimulatory effect of iron. However, relative Ag content and Ag(+)ion release were higher in low-Fe/Ag-EPS NPs than in high-Fe/Ag-EPS NPs, as revealed by emission-excitation spectra by luminescent spectrometry using a novel ad hoc established phycoerythrin fluorescence-based assay. Interestingly, high and low-Fe/Ag-EPS NPs showed different and growth medium-dependent minimal inhibitory concentrations againstStaphylococcus aureusATCC 29213 andPseudomonas aeruginosaATCC 15442. In addition, low-Fe/Ag-EPS NPs exert inhibition of staphylococcal and pseudomonal biofilm formation, while high-Fe/Ag-EPS NPs inhibits staphylococcal biofilm formation only. Altogether, these results, highlighting the different capability of Ag(+)release, support the idea that Fe/Ag-EPS NPs produced byK. oxytocaDSM 29614 can be considered as promising candidates in the development of specific antibacterial and anti-biofilm agents.