ABSTRACTMultidrug-resistant bacteria pose a tremendous challenge to public health worldwide. Many bacteria resistant to last-resort antibiotics due to antibiotic misuse have been recently reported, which may give rise to serious infections without effective treatment. Therefore, it is imperative to develop novel antimicrobial strategies. Natural phenols are known to increase bacterial membrane permeability and are potential candidates for the development of new antimicrobial agents. In this study, gold nanoparticles (Au NPs) carrying natural phenols were synthesized to combat bacteria resistant to last-resort antibiotics. Transmission electron microscopy, dynamic light scattering, zeta potential, and UV-visible spectra were used to characterize the synthesized Au NPs, which showed good monodispersity and uniform particle size. Evaluation of antibacterial activity using the broth microdilution method revealed that thymol-decorated gold nanoparticles (Thymol_Au NPs) had a broad antibacterial spectrum and higher bactericidal effects than last-resort antibiotics against last-resort-antibiotic-resistant bacteria. Considering the underlying antibacterial mechanism, the results showed that Thymol_Au NPs destroyed bacterial cell membranes. Further, Thymol_Au NPs were effective in treating mouse abdominal infections and exhibited acceptable biocompatibility without any significant toxicity in cell viability and histopathological assays, respectively, at most bactericidal concentrations. However, attention should be paid to changes in white blood cells, reticulocyte percentages, and superoxide dismutase activity during Thymol_Au NP treatment. In conclusion, Thymol_Au NPs have the potential for treating clinical infections caused by bacteria resistant to last-resort antibiotics.IMPORTANCEExcessive use of antibiotics can lead to bacterial resistance and the development of multidrug-resistant bacteria. Antibiotic misuse can also promote resistance against last-resort antibiotics. It is thus crucial to develop alternatives to antibiotics to retard the development of multidrug resistance. In recent years, the use of several nanodosage forms of antibacterial drugs has been investigated. These agents kill bacteria through a variety of mechanisms and avoid the problem of resistance. Among them, Au NPs, which are safer to use for medical applications than other metal nanoparticles, have attracted interest as potential antibacterial agents. To combat bacterial resistance to last-resort antibiotics and mitigate the problem of antimicrobial resistance, it is important and meaningful to develop antimicrobial agents based on Au NPs.