Photoluminescent gold nanodots (Au NDs) are prepared via etching and codeposition of hybridized ligands, an antimicrobial peptide (surfactin; SFT), and 1-dodecanethiol (DT), on gold nanoparticles (approximate to 3.2 nm). As-prepared ultrasmall SFT/DT-Au NDs (size approximate to 2.5 nm) are a highly efficient antimicrobial agent. The photoluminescence properties and stability as well as the antimicrobial activity of SFT/DT-Au NDs are highly dependent on the density of SFT on Au NDs. Relative to SFT, SFT/DT-Au NDs exhibit greater antimicrobial activity, not only to nonmultidrug-resistant bacteria but also to the multidrug-resistant bacteria. The minimal inhibitory concentration values of SFT/DT-Au NDs are much lower (>80-fold) than that of SFT. The antimicrobial activity of SFT/DT-Au NDs is mainly due to the synergistic effect of SFT and DT-Au NDs on the disruption of the bacterial membrane. In vitro cytotoxicity and hemolysis analyses have revealed superior biocompatibility of SFT/DT-Au NDs than that of SFT. Moreover, in vivo methicillin-resistant S. aureus-infected wound healing studies in rats show faster healing, better epithelialization, and are more efficient in the production of collagen fibers when SFT/DT-Au NDs are used as a dressing material. This study suggests that the SFT/DT-Au NDs are a promising antimicrobial candidate for preclinical applications in treating wounds and skin infections.