The AmB-Cu(II) complex has recently been reported as an antifungal agent with reduced aggregation of AmB in aqueous solutions, increased anti C. albicans activity and lower toxicity against human cells in vitro. In the present work, investigations of the activity of the AmB-Cu (II) complex against fungal pathogens with varying susceptibility, including C. albicans and C. parapsilosis strains and intrinsically resistant A. niger, and cytotoxicity in normal human dermal fibroblasts (NHDF) in vitro were performed. For better understanding of the mechanism of reduced cytotoxicity and increased fungicidal activity, the influence of the AmB-Cu (II) complex on membrane integrity and accumulation of cellular reactive oxygen species (ROS) and mitochondrial superoxide was compared with that of conventional AmB. In the sensitive C. albicans and C. parapsilosis strains, the AmB-Cu(II) complex showed higher fungicidal activity (the MIC value was 0.35–0.7 μg/ml for the AmB-Cu (II) complex, and 0.45–0.9 μg/ml for Fungizone) due to increased induction of oxidative damage with rapid inhibition of the ability to reduce tetrazolium dye (MTT). In the NHDF cell line, the CC50 value was 30.13 ± 1.53 μg/ml for the AmB-Cu(II) complex and 17.46 ± 1.24 μg/ml for (Fungizone), therefore, the therapeutic index (CC50/MIC90) determined in vitro was 86.09–43.04 for the AmB-Cu(II) complex and 38.80–19.40 for Fungizone. The lower cytotoxicity of the AmB-Cu(II) complex in human cells resulted from lower accumulation of cellular and mitochondrial reactive oxygen species. This phenomenon was probably caused by the induction of successful antioxidant defense of the cells. The mechanism of the reduced cytotoxicity of the AmB-Cu(II) complex needs further investigation, but the preliminary results are very promising.