Asparagine is one of the precursors of acrylamide and toxic fungal secondary metabolites, both carcinogenic compounds. In the present study, the optimal conditions to deplete asparagine by Aureobasidium pullulans (L1 and L8) from potato and wheat flour matrices were investigated. Through a colorimetric plate-assay with phenol red as indicator dye, both strains demonstrated to be able to produce L-asparaginase from 20 {}^{\circ }C to 30 {}^{\circ }C for L1 and only at 20 {}^{\circ }C for L8 strain starting from 48 h of incubation. The ability of both yeasts to reduce asparagine content in potato and wheat flour was studied by in vitro spectrophotometric assay. Both strains showed a great ability to totally reduce asparagine at 20 {}^{\circ }C after 15 min of incubation in potato homogenate, conversely in wheat flour, the highest reduction was detected after a longer exposition time (60 min). As known, L1 and L8 diamine asparaginase to aspartic acid. For this reason, both amino acids were tested to verify the antifungal effect against Rhizoctonia solani (Rs1) and Fusarium graminearum (F3) mycelial growth. Asparagine (120 mg/L) increased Rs1 and F3 mycelial growth respectively by 4.4% and 18.9%; conversely, aspartic acid significantly inhibited both respectively by 8.2% and 12.0%.