Room-temperature storage and transportation of microorganisms maximize the power of microorganisms in healthcare, energy, and environment. Recently, paper-based biotechnologies have been developed to enable room-temperature storage of a variety of nonliving biosystems such as diagnostic devices and cell-free systems. Herein, room-temperature storage of living microorganisms is realized by an electrospun nonwoven paper containing convex region, which is composed of coiled microfibers with dense distribution of microorganisms. Microorganisms are encapsulated into the microfibers and remain intact after electrospinning. Poly(ethylene oxide) is used as polymer matrix, and glycerol and dextran are used as additives. When the contents of glycerol and dextran are optimized as 5 and 0.4%, the room-temperature time is prolonged to 2 days, more than 8 folds as compared with the control group. Upon demand, the microorganisms can be activated by adding water and used for culturing microorganisms directly. Furthermore, mechanisms which account for microbial activity and storage are studied. Our microfiber-based strategy is universal for the room-temperature storage of prokaryotic and eukaryotic microorganisms in the solid formulation. Besides, our microorganism/polymer complex structures represent novel living materials via a bottom-up strategy, which are of great potential for new biomedical applications.