The anti-biofouling and desalination properties of thin film composite reverse osmosis membranes (TFC-RO), modified by the incorporation of copper and iron nanoparticles, were compared. Nanoparticles of metallic copper (CuNPs) and an iron crystalline phase mix (Fe and Fe2O3, FeNPs) were obtained by oxide-reduction-precipitation and reduction reactions, respectively, and characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM) techniques. Modified membranes (PA+0.25Cu-PSL and PA+0.25Fe-PSL) were obtained by incorporating these nanoparticles during the interfacial polymerization process (PI). These membranes were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), and contact angle measurements. Bactericidal tests by a Colony Forming Unit (CFU) were performed using Escherichia coli, and anti-adhesion properties were confirmed by fluorescence microscopy estimating the percentage of live/dead cells. The permeate flow and rejection of salts was evaluated using a crossflow cell. An increase of the membrane’s roughness on the modified membrane was observed, influencing the desalination performance more strongly in the presence of the FeNPs with respect to the CuNPs. Moreover, a significant bactericidal and anti-adhesion effect was obtained in presence of both modifications with respect to the pristine membrane. An important decrease in CFU in the presence of modified membranes of around 98% in both modifications was observed. However, the anti-adhesion percentage and reduction of live/dead cells were higher in the presence of the copper-modified membrane in comparison to the iron-modified membrane. These facts were attributed to the differences in antimicrobial action mechanism of these types of nanoparticles. In conclusion, TFC-RO membranes modified by the incorporation of CuNPs during PI represent one alternative material to attend to the biofouling impact in the desalination process.