In this study, we used sol-gel to synthesize undoped and V-ZnO nanoparticles with different vanadium concentrations (1, 3, and 5 at.%) under supercritical dry conditions of ethanol. XRD spectra showed that the obtained powders are well crystallized in the hexagonal wurtzite structure of ZnO nanoparticles. The average crystallite size, estimated by the Debye-Scherer formula, was found to be equal to 31 nm for the pure sample, and it was decreased to 27 nm for the 3at.% vanadium-doped one. SEM and TEM photographs indicated the spherical and elongated shapes of the nanoparticles. The stretching bands located at 419 cm−1 confirmed ZnO material formation. The efficacy of the produced ZnO NPs against Gram+, Gram− bacteria, and fungi was tested. Vanadium-doped ZnO, with low concentrations (10 µg/mL), exhibited a large influence on bacterial and fungi growth inhibition. For example, the inhibition zones IZ of S. aureus and E. coli bacteria reached 16 and 15 mm, respectively, for ZnO:V1%, while the IZ of these two bacteria were 14 and 12 mm for the undoped ZnO. The use of V-dopant enhanced the production of the reactive oxygen species ROS by the photogeneration of electron-hole pairs due to light absorption by ZnO in the visible region.