Biological particles in heritage-related indoor environments (museums, libraries, archives) represent a hazard to artifacts (biodeteriogenic action), operators and visitors’ health. The aim of the study was to evaluate environmental biological contamination and microclimate conditions in different periods of the year in De Rossi room of the historical Palatina Library in Parma. Microclimatic measurements were recorded continuously for a period ranging from 11 to 17 days in: January–February, May and September 2017. Monitoring of bacterial and fungal contamination was performed for air by active and passive sampling and by nitrocellulose membranes on shelves and manuscripts. Microorganisms were isolated by in vitro culture (Tryptic Soy Agar and Sabouraud Dextrose Agar) and characterized by molecular investigation. Viable and non-viable fungal spores were collected by Hirst spore trap. Concerning air, the highest bacterial mean values were 76.67 cfu/m3 (in May) and 3.33 IMA (in February, May and September), while for fungi 60.67 cfu/m3 and 4.33 IMA in September. The highest fungal values, both on shelves and books, were recorded in September with Alternaria, Arthrinium, Aspergillus, Cladosporium and Penicillium spp. being the mainly isolated fungi. Air temperature, relative humidity and air velocity reached mean values ranging, respectively, from 14.71 to 26.60 °C, from 41.62 to 44.83% and from 0 to 0.04 m/s. This case study provides an assessment of the environmental quality over a long period, representing a reference model to better understanding microbiological contamination of cultural heritage environment toward the improvement of artwork conservation strategies and the safeguard of human health.