One-pot synthesis of cuprous ferrite (CuFeO2) nanoparticles modified with glycerol (glycerol@CuFeO2 NPs) using oligol and reducing agent was reported. The antifungal activities of the nanoparticles for Candida utilis using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) are also reported. Glycerol@CuFeO2 NPs are synthesized via hydrothermal method and characterized using TEM, SEM, EDX, XRD, FTIR, DLS, and zeta potential. The data revealed that glycerol@CuFeO2 NPs show an efficient ability to degrade contaminants, such as fungi in the aquatic environment. The proteomic analysis of the pathogenic fungus upon addition of different concentrations of glycerol@CuFeO2 NPs and different incubation times reveals the degradation of high molecular weight protein to small proteins or peptides.