When natural regeneration of Quercus robur stands is hampered by an insufficient acorn yield, human assisted sowing of acorns collected in non-affected stands and stored for some period of time is performed. To inhibit the development of fungi and acorn deterioration during storage, thermotherapy is usually applied by submerging acorns for 2.5 h in water heated to 41 °C. This research aimed to test the effect of four thermotherapy treatments of different durations and/or applied temperatures as well as short-term storage at −1 °C or 3 °C on acorn internal mycobiota and germination. Fungal presence in cotyledons was analyzed in 450 acorns by isolation of mycelia on artificial media, followed by a DNA-based identification. Germination of 2000 acorns was monitored in an open field trial. Thermotherapy significantly decreased fungal diversity, while storage at 3 °C increased the isolation frequency of several fungi, mainly Penicillium spp. The most frequently isolated fungi did not show a negative impact on acorn germination after short-term storage. The study confirmed the efficiency of thermotherapy in the eradication of a part of acorn internal mycobiota, but also its effect on the proliferation of fast-colonizing fungi during storage. However, the latter showed to be more stimulated by storage conditions, specifically by storage at 3 °C.