Proteases play a key role in cell defense mechanisms to cold-induced oxidative stress. Data on the relationship between cold stress, growth phase, and temperature preferences of the fungal strains isolated from different habitats are very scarce. Here, we report changes in the intra- and extracellular protease activity of three fungal Penicillium strains (two Antarctic and one temperate) under transient temperature downshift during exponential- and stationary growth phases. The results indicated enhanced enzyme levels in both growth phases depending on the degree of stress and strain thermal class. In order to explain the obtained data, we compared them with our previous results on the protein carbonyl content, accumulation of oxidative-stress biomarkers, and antioxidant enzyme defense in the same three fungal strains. The cell response was affected by the temperature preference of the strain, but not by the climatic distance between the locations of isolation.