ABSTRACT Therapeutic options to treat bacterial infections caused by Gram-negative pathogens are limited due to the spread of multidrug resistance. Protein engineering of phage-derived lysins can play a key role in the search for new antimicrobial compounds targeting Gram-negative pathogens. A previous high-throughput screen of a combinatorial lysin library identified lysin 1D10 active against Acinetobacter baumannii under elevated human serum concentrations. The engineered lysin consists of four modules: cecropin A, a linker, cell wall-binding domain, and an enzymatic active domain. Using time-lapse microscopy, we show that 1D10 has a distinct antibacterial mode of action resulting in local cell wall bulging at the septum instead of cell-wide lysis, as observed for previously reported engineered lysins that target Gram-negative bacteria. Our results indicate that the activity of 1D10 relies on the antibacterial activity of both cecropin A (CecA) and the enzymatically active domain. Based on a truncation analysis, the role of each of the four modules of 1D10 was dissected. We further compared the antibacterial spectrum, thermostability, and cytotoxicity of cecropin A alone and lysin 1D10. Both lysin 1D10 and CecA are most active against A. baumannii and are not cytotoxic toward human keratinocytes. Lysin 1D10 unfolds at 57°C and has a remarkable refolding capacity, as it regains its activity even after exposure to 90°C and sterilization conditions, whereas CecA is inactivated at 70°C. Overall, the present study shows that an improved understanding of the killing mechanism and the protein properties will further support lysin engineering designs in the future. IMPORTANCE Engineered lysins are considered as highly promising alternatives for antibiotics. Our previous screening study using VersaTile technology identified 1D10 as a possible lead compound with activity against Acinetobacter baumannii strains under elevated human serum concentrations. In this manuscript, we reveal an unexpected mode of action and exceptional thermoresistance for lysin 1D10. Our findings shed new light on the development of engineered lysins, providing valuable insights for future research in this field.