One of the largest nursery colonies of Rhinolophus ferrumequinum in Italy occurs in the Natural Park of Migliarino San Rossore Massaciuccoli, Tuscany, with the species roosting in several buildings. We identified various roosts used by R. ferrumequinum within the park and investigated their microclimatic parameters (temperature, relative humidity and daily temperature excursion). We monitored roost use for one year and found it not only to be influenced by season and the microclimatic conditions of different roosts, but also by occasional stress conditions such as disturbance by predators. We also studied nightly emergence behavior, specifically we considered how this is influenced by climatic (temperature, relative humidity and evening light intensity) and demographic (presence of pups, subadults and colony size) parameters. The colony always performed a pre-emergence behavior and we found that its onset occurred earlier at higher temperatures, lower evening light intensity, in larger colonies and in the presence of pups. High temperature and low evening light intensity also resulted in a longer nightly emergence, which was also identified in larger colonies and when subadults were present. This study highlights the importance of the conservation of multiple roosts within the distribution range of R. ferrumequinum nurseries. Additionally, we demonstrated how certain climatic and demographic factors influence both pre-emergence and emergence behavior.