Biomass degradation by microorganisms may cause major losses during the storage of wood chips for energy production. Poplar wood chips from short rotation coppices are especially prone to degradation with dry matter losses (DML) of up to 25% within a storage period, emphasizing the need for countermeasures. Therefore, we investigated the potential of the addition of alkaline Ca(OH)2 to the wet biomass of poplar wood chips and hypothesised that the establishment of an alkaline environment would reduce the activity of fungi, the primary wood degraders. Three industrial-scale piles (250 m³) with 0, 1.5 and 3% Ca(OH)2 were installed in Güssing, Austria and for four months (April–August 2019) the pile temperature, pH, moisture content, gas evolution (O2, CO2, H2, H2S, CH4) as well as DML were monitored. Ca(OH)2 altered the physicochemical properties of the wood chips but did not prevent biomass losses. However, as compared to literature, the DML were, compared to earlier investigations, also low in the control. In addition, cultivation methods were performed to evaluate the diversity of thermophilic microbes throughout the storage. Numerous filamentous fungi belonging to the phyla Ascomycota and Mucoromycota were isolated, being Rhizomucor pusillus, Aspergillus fumigatus, Thermomyces lanuginosa and Thermoascus aurantiacus the dominant species. Only minor differences in the fungal composition were detected as a result of Ca(OH)2 addition. Instead, clear shifts in colony forming units (CFUs) were detected as a function of progressing storage time, with a decrease of the number of propagules after four months.