In the laboratory, non-chlorophyllic (orthodox) fern spores are long-lived. However, their potential longevity might not reflect ecological longevity, which determines their viability within the natural soil spore bank. Our aim was to compare ecological (in a semi-natural soil bank) and potential (storage in laboratory–suboptimal conditions) longevity and vigour of fern spores over a period of five months. We assessed these in Llavea cordifolia Lag., Pellaea ovata (Desv.) Weath., and Thelypteris ovata R.P. St. John; spores collected from a cloud forest in Hidalgo, Mexico. The spore samples were enclosed in permeable phytoplankton bags and buried in situ at 10 cm soil depth, whereas controls were stored in glass vials in the laboratory at room temperature (21.6 ± 1.8°C). After one, three, and five months, the spores of both treatments were germinated, and the final germination percentage and vigour traits (lag time, germination rate, and mean germination time) were evaluated. After five months, the spore viability of three species remained high under both storage conditions but their vigour was affected differentially. The soil-stored spores of L. cordifolia remained more vigorous and had a shorter lag and mean germination time than laboratory-stored spores. In the other two species, spore vigour declined in both storage treatments, reflecting in longer lag and mean germination time and lower germination rate. In all three species, the moist soil environment of the cloud forest maintained spore viability for over five months. However, the decline of spore vigour might be disadvantageous when competing for resources during later stages of colonization. Our study is a first experimental approach to understand the time-effect of soil storage on viability and vigour of fern spores when compared to laboratory storage. Long-term studies in different soils, climates and vegetation types are necessary to determine spore ecological longevity of different fern species.