Desiccation conditions and storage temperature are crucial factors for the successful long-term storage of fern spores. This study aimed to determine the optimal desiccation conditions and storage temperatures for the long-term survival of eastern bracken (Pteridium aquilinum var. latiusculum) spores. The effect of different storage temperatures (25°C, 4°C, −20°C, −80°C, and −196°C for up to 48 weeks) on spore germination and sporophyte development was investigated in collected spores. The germination percentage remained constant in all spores, except in those stored at −20°C and 25°C. Spores stored at −20°C had the lowest germination percentage and were almost completely nonviable. The collected spores were desiccated for one week at various relative humidity (RH) levels equilibrated using saturated salt solutions. The water content was measured in desiccated spores, and the spores were stored in liquid nitrogen for up to 48 weeks. The water content of the spores desiccated at 15–55% RH was 5.0–5.4%, and their germination percentage remained constant with storage time. Desiccation at 94% RH increased the spore water content to 9.5% and decreased the germination percentage. The relatively high water content in spores was not suitable for their long-term storage. Long-term survival of the spores can be achieved by desiccating the spores at low RH to adjust the spore water content to 5% prior to their storage at low temperature.