Introduction: Bacterial resistance is a worldwide public health problem, requiring new therapeutic options. An alternative approach to this problem is the use of animal toxins, such as phospholipases (PLA₂) isolated from snake venom, which have important biological activities. Bothrops erythromelas is one of the snake species in the Northeast of Brazil that attracts great medical-scientific interest. Here we aimed to purify and characterize a PLA₂ from B. erythromelas, searching for heterologous activities against bacterial biofilm. Methods: Venom extraction and quantification were followed by RP-HPLC in C18 column, MALDI-ToF mass spectrometry and sequencing by Edman degradation. All experiments were monitored by specific activity using 4-nitro-3 (octanoyloxy) benzoic acid (4N₃OBA) substrate. In addition, hemolytic tests and anti-bacterial tests including action against Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii, were carried out. Moreover, tests of antibiofilm action against A. baumannii were also performed. Results: PLA₂, after one purification step, presented 31 N-terminal amino acid residues, and molecular weight of 13656.4 Da with enzymatic activity confirmed in 0.06 µM concentration. Antibacterial activity against S. aureus (IC₅₀ = 30.2 µM) and antibiofilm activity against A. baumannii (IC₅₀ = 1.1 µM) were observed. Conclusions: This is the first time that PLA₂ purified from B. erythromelas venom has appeared as an alternative candidate in studies of new antibacterial medicines.