Composting is a complex process in which various micro-organisms, mainly fungi and bacteria, are involved. The process depends on a large number of factors (biological, chemical, and physical) among which microbial populations play a fundamental role. The high temperatures that occur during the composting process indicate the presence of thermotolerant and thermophilic micro-organisms that are key for the optimization of the process. However, the same micro-organisms can be harmful (allergenic, pathogenic) for workers that handle large quantities of material in the plant, and for end users, for example, in the indoor environment (e.g., pots in houses and offices). Accurate knowledge of thermotolerant and thermophilic organisms present during the composting stages is required to find key organisms to improve the process and estimate potential health risks. The objective of the present work was to study thermotolerant and thermophilic mycobiota at different time points of compost maturation. Fungi were isolated at four temperatures (25, 37, 45, and 50 °C) from compost samples collected at five different steps during a 21-day compost-maturation period in an active composting plant in Liguria (northwestern Italy). The samples were subsequently plated on three different media. Our results showed a high presence of fungi with an order of magnitude ranging from 1 × 10 4 to 3 × 10 5 colony-forming units (CFU) g −1. The isolated strains, identified by means of specific molecular tools (ITS, beta-tubulin, calmodulin, elongation factor 1-alpha, and LSU sequencing), belonged to 45 different species. Several thermophilic species belonging to genera Thermoascus and Thermomyces were detected, which could be key during composting. Moreover, the presence of several potentially harmful fungal species, such as Aspergillus fumigatus, A. terreus, and Scedosporium apiospermum, were found during the whole process, including the final product. Results highlighted the importance of surveying the mycobiota involved in the composting process in order to: (i) find solutions to improve efficiency and (ii) reduce health risks.