Microorganisms are one of the most attractive and simple sources for the synthesis of different types of metal nanoparticles. The synthesis of manganese dioxide nanoparticles (MnO2 NPs) by microorganisms from reducing potassium permanganate was investigated for the first time in the present study. The microbial supernatants of the bacterium Saccharophagus degradans ATCC 43961 (Sde 2-40) and of the yeast Saccharomyces cerevisiae showed positive reactions to the synthesis of MnO2 NPs by displaying a change of color in the permanganate solution from purple to yellow. KMnO4-specific peaks also disappeared and MnO2-specific peaks emerged at an absorption maximum of 365 nm in UV-visible spectrophotometry. The washed Sde 2-40 cells did not show any ability to synthesize MnO2 NPs. The medium and medium constituents of Sde 2-40 showed similar positive reactions as supernatants, which indicate the role of the Sde 2-40 medium constituents in the synthesis of MnO2 NPs. This suggests that microorganisms without nanoparticle synthesis ability can be misreported for their abilities to synthesize nanoparticles. S. cerevisiae washed cells showed an ability to synthesize MnO2 NPs. The strategies of keeping yeast cells in tea bags and dialysis membranes showed positive tests for the synthesis of MnO2 NPs. A Fourier transform-infrared spectroscopy study suggested roles for the proteins, alcoholic compounds, and cell walls of S. cerevisiae cells in the synthesis of MnO2 NPs. Electron-dispersive X-ray spectroscopy analyses confirmed the presence of Mn and O in the sample. X-ray photoelectron spectroscopy revealed characteristic binding energies for MnO2 NPs. Transmission electron microscopy micrographs revealed the presence of uniformly dispersed hexagonal- and spherical-shaped particles with an average size of 34.4 nm. The synthesis approach using yeast is possible by a simple reaction at low temperature without any need for catalysts, templates, or expensive and precise equipment. Therefore, this study will be useful for the easy, cost-effective, reliable, and eco-friendly production of nanomaterials.