Survival and functioning of mycorrhizal associations at low temperatures are not known well. In an earlier study, ectomycorrhizas did not affect the frost hardiness of Scots pine (Pinus sylvestris L.) roots, but here we studied whether differential nutrient availability would change the result and additionally, alter frost hardiness aboveground. The aim in this experiment was to compare the frost hardiness of roots and needles of mycorrhizal (Hebeloma sp.) and non-mycorrhizal Scots pine seedlings raised using two fertilization treatments and two cold-hardening regimes. The fertilization treatments were low (LF) and high (HF) application of a complete nutrient solution. Three hundred mycorrhizal and non-mycorrhizal seedlings were cultivated in growth chambers in four blocks for 16 weeks. For the first 9 weeks, the seedlings grew in long-day and high-temperature (LDHT) with low fertilization and then they were raised for 3 weeks in LDHT with either low or high fertilization. After this, half of the plants in each treatment combination remained in LDHT, and half were transferred to short-day and low-temperature (SDLT) conditions to cold acclimatize. The frost hardiness of the roots and needles was assessed using controlled freezing tests followed by electrolyte leakage tests (REL). Mycorrhizal roots were slightly more frost hardy than non-mycorrhizal roots, but only in the growing-season conditions (LDHT) in low-nutrient treatment. In LDHT and LF, the frost hardiness of the non-mycorrhizal roots was about -9 °C, and that of the non-mycorrhizal HF roots and the mycorrhizal roots in both fertilization levels was about -11 °C. However, no difference was found in the roots within the SDLT regime, and in needles, there was no difference between mycorrhizal and fertilization treatments. The frost hardiness of needles increased by SDLT treatment, being -8.5 and -14.1 °C in LDHT and SDLT, respectively. The dry mass of roots, stems, and needles was lower in LF than in HF and lower in SDLT than in LDHT. Mycorrhizal treatment did not affect the dry mass or its allocation. Although the mycorrhizal roots were slightly more frost hardy in the growing-season conditions, this is not likely to have significance in the field.