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Summary: The emergence and rapid spread of multidrug‐resistant bacteria has induced intense research for novel therapeutic approaches. In this study, the Acinetobacter baumannii bacteriophage D2 (vB_AbaP_D2) was isolated, characterized and sequenced. The endolysin of bacteriophage D2, namely Abtn‐4, contains an amphipathic helix and was found to have activity against multidrug‐resistant Gram‐negative strains. By more than 3 log units, A. baumannii were killed by Abtn‐4 (5 µM) in 2 h. In absence of outer membrane permeabilizers, Abtn‐4 exhibited broad antimicrobial activity against several Gram‐positive and Gram‐negative bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterococcus and Salmonella. Furthermore, Abtn‐4 had the ability to reduce biofilm formation. Interestingly, Abtn‐4 showed antimicrobial activity against phage‐resistant bacterial mutants. Based on these results, endolysin Abtn‐4 may be a promising candidate therapeutic agent for multidrug‐resistant bacterial infections. [ABSTRACT FROM AUTHOR]