The l-amino acid oxidase from Vipera lebetina venom was purified to homogeneity using combination of size exclusion, ion exchange and hydrophobic chromatography. The monomeric molecular mass of the homodimeric enzyme is 60.9 kDa. The N-terminal and the tryptic peptides share high homology with other snake venom l-amino acid oxidases. The enzyme displays high specificity towards hydrophobic l-amino acids, the best substrates are l-Met, l-Trp, l-Leu followed by l-His, l-Phe, l-Arg and l-Ile. Six substrates—Gly, l-Ser, l-Thr, l-Pro, l-Cys, l-Asp—were not oxidized. The enzyme has antimicrobial activity inhibiting the growth of both Gram-negative and Gram-positive bacteria. V. lebetina LAAO dose-dependently inhibited platelet aggregation induced by ADP or collagen. In case of ADP-induced aggregation the inhibitory effect was more pronounced on the second wave of aggregation.