Fungal aerosols deteriorate library collections and can impact human health, mainly via respiratory diseases. Their spread is influenced by factors such as temperature and relative humidity. This study aims to assess the concentration of fungal aerosols in the interior environment of the Popular Library of Gaira in the District of Santa Marta, Colombia, using a two-stage cascade impactor utilizing Sabouraud dextrose agar on Petri dishes for the collection of samples. The sampler was positioned at 1.5 m above ground level, operated with a flow rate of 28.3 l/min for 4 min and thermo-hygrometric conditions were also recorded. Concentrations in the air of up to 1197.0 CFU/m3 were reported, with a mean value close to 150 CFU/m3. Higher values during the morning samples were noted. Seven genera of fungi were found, Aspergillus and Curvularia were the most abundant. The temperature was between 30.80 and 33.51 °C, and the relative humidity was between 62.61 and 64.80%. Statistical analysis showed a significant correlation between the fungal aerosol concentration and relative humidity, where an increase of 10% in moisture could double the fungal aerosol concentration. We concluded that potentially favorable conditions exist indoors for the growth and survival of the following fungi: Aspergillus, Penicillium, Cladosporium, and Curvularia, and to a lesser extent for Chrysonilia, Cunninghamella, and Paecylomices. Relative humidity was seen to be the factor that affects the concentration of aerosols fungal in the library most significantly.