OBJECTIVE: In this study, we aimed to develop new Lipo-niosomes based nanoparticles loaded with Amphotericin B (AmB) and Thymus Essential Oil (TEO) and test their effectiveness in the treatment of fungal-infected human adipose stem cells (hASCs). MATERIALS AND METHODS: In this experimental study, optimal formulation of AmB and TEO loaded lipo-niosome (based on lipid-surfactant thin-film hydration method) was chemically, and biologically characterized. Therefore, encapsulation capacity, drug release, size, and the survival rate of cells with different concentrations of free and encapsulated AmB/ TEO were evaluated using the MTT method, and its antifungal activity was compared with conventional AmB. RESULTS: Lipo-Niosome containing Tween 60 surfactant: cholesterol: Dipalmitoyl phosphatidylcholine (DPPC): Polyethylene glycol (PEG) with a ratio of 20:40:60:3 were chosen as optimal formulation. Lipo-Niosomes entrapment efficiency was 94.15%. The drug release rate after 24 hours was 52%, 54%, and 48% for Lipo-AmB, Lipo-TEO, and Lipo-AmB/TEO, respectively. Physical and chemical characteristics of the Lipo-Niosomes particles indicated size of 200 nm and a dispersion index of 0.32 with a Zeta potential of -24.56 mv. Furthermore, no chemical interaction between drugs and nano-carriers was observed. The cell viability of adipose mesenchymal stem cells exposed to 50 μg/ml of free AmB, free TEO, and free AmB/TEO was 13.4, 58, and 36.9%, respectively. Whereas the toxicity of the encapsulated formulas of these drugs was 48.9, 70.8, and 58.3% respectively. The toxicity of nanoparticles was very low (8.5%) at this concentration. Fluorescence microscopic images showed that the antifungal activity of Lipo-AmB/ TEO was significantly higher than free formulas of AmB, TEO, and AmB/TEO. CONCLUSION: In this study, we investigated the efficacy of the TEO/AmB combination, in both free and encapsulated- niosomal form, on the growth of fungal infected-hASCs. The results showed that the AmB/TEO-loaded Lipo-Niosomes can be suggested as a new efficient anti-fungal nano-system for patients treated with hASCs