In this research, the in vitro antimicrobial effect of zinc oxide (ZnO), copper oxide (CuO) and iron oxide (Fe2O3) nanoparticles (NPs)—with average sizes of 20, 46 and 30 nm, respectively—on the root rot disease caused by the fungus Fusarium oxysporum and on blight disease caused by the fungus Alternaria solani were studied. Also, bacterial diseases caused by Clavibacter michiganensis and Pseudomonas syringae that infects a wide range of plant species were assessed. Different concentrations of NPs (0, 100, 250, 500, 700 and 1,000 mg/L) were prepared on PDA agar or King's B medium in a complete randomized design with four replicates. According to the results, ZnO NPs exhibited an outstanding inhibitory effect against fungi and bacteria strains. The above results were associated with the smaller particle size. Fungi strains showed a differential sensitivity depending on the kind of NPs used. A. solani showed the highest sensitivity to ZnO NPs at 1,000 mg/L (99%), followed by CuO NPs at the same dose (95%). Fe2O3 NPs at all evaluated doses had no inhibitory effects on the mycelia growth of this strain, although F. oxysporum revealed greater effectiveness of the CuO NPs (96%) compared with ZnO NPs since it only inhibited 91% of the mycelial growth. The antibacterial activity was studied through optical density. C. michiganensis was found to be more sensitive to ZnO NPs because a lesser dose (700 mg/L) was required to reduce the bacterial growth (90%); in comparison, P. syringae required a dose of 1,000 mg/L to inhibit its growth (67%). CuO NPs displayed the smallest growth inhibition against the bacteria strains analysed. The antimicrobial effect of the metallic NPs that were assayed increased with higher doses.