The design route, synthesis, and characterization of spherical copper nanoparticles with antifungal potential are reported in the present work. Copper nanoparticles were synthesized by a novel, inexpensive, and eco‐friendly chemical reduction method using ascorbic acid as a reductant and stabilizer under reflux conditions. The characterization results showed the formation of homogeneous, dispersed, and stable spherical ascorbic acid‐capped copper nanoparticles (CuNPs) with a diameter of 250 nm. The CuNPs exhibited sustained antifungal activity against Candida albicans (C. albicans) after 24 h and even 48 h of incubation. Using enhanced dark‐field microscopy, we presented the in situ interaction between CuNPs and C. albicans. Here, part of the interaction of CuNPs among the C. albicans, studied without the use of any chemical and/or physical fixing method, is discussed. The results indicate that part of the antifungal mechanism involves a promoted adhesion of CuNPs onto the cell wall and a massive accumulation of CuNPs into the fungal cells, concluding in cellular leakage. The cytotoxicity (viability) evaluations indicated that our CuNPs were more biocompatible after comparison to the Cu precursor and triclosan (a commercial antifungal drug). The synthesized CuNPs will open up a new road for their possible use as a potent antimicrobial agent for clinical and industrial applications.