Two parameters of a one step freeze-drying process, cooling rate and protecting media, are studied in an effort to improve the viability and the stability of the preserved fungal strains. Cooling rates of 1.6 C, 3 C and 40 C/min are applied on cells of Saccharomyces cerevisiae and Brettanomyces bruxellensis and on spores of Trichoderma viride and Arthrobotrys arthrobotryoides preserved in 93 suspending media containing polymers, sugars, albumin, milk, honey, polyols, amino acids alone or in combination. The viability rate of Saccharomyces cerevisiae cells is increased from 30% to 96-98% by using an appropriate protecting medium containing 10% skim milk with 2 compounds among honey, sodium glutamate, trehalose or raffinose in the freeze-drying process carried out at a 3 C/min cooling rate. In the same conditions Arthrobotrys arthrobotryoides spores, the most sensitive strain among the four tested, provides 60-65% viability, while this strain does not survive a classical freeze-drying in 10% skim milk. Moreover, with the improved method the stability of properties of Penicillium expansum is demonstrated.