Trichophyton rubrum (T.rubrum) is a pathogenic dermatophyte that can causes fungal infection in keratinized layer of the human tissues such as skin, hair and nails. This work was carried out to study the antifungal activity of CuO nanoparticles (CuO NPs) on the viability of T.rubrum. CuO NPs have been synthesized by pulsed laser ablation of copper target immersed in liquid media using Q-switched pulsed Nd:YAG laser with 1064 nm. The optical properties and the surface charge of CuO NPs colloidal were characterized using UV–Vis spectrophotometer and Zeta potential techniques. UV–Vis spectrophotometer exhibited two peaks of absorption of CuO NPs colloidal: sharp peak at 200 nm and another peak at 630 nm. Zeta potential technique showed negative charge of CuO NPs colloidal (-28.16 mV). The morphological properties of CuO NPs such as particle size, shape and particle size distribution were characterized using Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM), the shapes were spherical and the particle size distribution was inhomogeneous which ranges between 20 to 180 nm. The synthesized CuO NPs presented suitable fungistatic activity against T.rubrum and its highest growth-inhibitory effectiveness was at high concentration (100 μg/ml) and high exposure time (3 hrs). Moreover, the inhibition rate of T. rubrum progressively increased with increasing CuO NPs concentration and exposure time.