AbstractDespite the advances in technology, bacterial infection associated with biomedical devices is still one of the most challenging issues in clinical practice. Incorporation of antimicrobial agents is regarded as an efficient way to combat medical device associated infectious. However, most of antimicrobial agents have high toxicity to host cells. Thus, fabrication of novel antimicrobial agents that simultaneously fulfill the requirements of antibacterial activity as well as biocompatibility is urgently needed. Herein, a series of water‐insoluble antibacterial complexes based on hyperbranched poly‐L‐lysine (HBPL) and four different surfactants through non‐covalent interactions are developed. Such kinds of surfactants have great effects on the antibacterial property of poly(ɛ‐caprolactone) (PCL) films that incorporate with the HBPL‐based complexes. The results reveal that the PCL films that doped with HBPL/phosphate ester surfactant complexes showed the highest bacterial killing efficiency. Moreover, the cytocompatibility of the composite films is also investigated. Hemolysis experiments indicate that all the films had low hemolytic activities. Considering the excellent antimicrobial and cytocompatibility properties, this work believes that the optimized complexes have great potential to be used as antimicrobial agents in biomedical field.