We report the detailed molecular characterization of two PLA₂s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA₂s, named BlK-PLA₂ and BlD-PLA₂, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA₂s, and show a high degree of sequence similarity to homologous snake venom PLA₂s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA₂s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA₂s were assessed by quantitative Real-Time PCR. Bl-PLA₂s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA₂s as tools of research.