The family Ophioglossaceae is one of the oldest fern lineages, characterized by a specific sporophyte structure where each leaf is divided into a fertile (sporophore) and a sterile (trophophore) segment. The aims of this study were to analyze: (1) the growth rate and duration for each developmental stage of the sporophyte of the protected fern Botrychium lunaria (L.) Sw., (2) the correlation between sporophore and trophophore size, and (3) the effects of soil conditions and density of other herbaceous plants on the development and number of individuals of B. lunaria to suggest some possible methods for its protection. Field research was conducted in a threatened population of this species in alluvial ash forest (Astrantio-Fraxinetum Oberd. 1953) at the base of Dziewicza Góra, a wooded hill in western Poland. During leaf emergence, the first and longest stage of development, the leaf developed intensively. Subsequently, its subdivision into the sporophore and trophophore became apparent. Throughout the four subsequent stages of sporophyte development (initial maturation, final maturation, spore release and senescence), the sizes of the trophophore and sporophore were significantly correlated. Additionally, sporophore size was affected by abiotic factors, including the total N and organic C contents of the soil. In patches with a higher cover of the herb layer we observed a smaller number of individuals of B. lunaria, so active protection by control of competing plant species seems necessary to ensure the survival of this population. Our results may help to design an effective conservation strategy for this rare and threatened fern in Poland and elsewhere.