The root of Carlina acaulis L. has been widely used in traditional medicine for its antimicrobial properties. In this study, the fractionation of methanol extract from the root was conducted. Four fractions (A, B, C, and D) were obtained and tested against a range of bacteria and fungi. The results showed promising antibacterial activity, especially against Bacillus cereus, where the minimal inhibitory concentration (MIC) was determined to be equal to 0.08 mg/mL and 0.16 mg/mL for heptane (fraction B) and ethyl acetate (fraction C), respectively. In the case of the methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 strain, the same fractions yielded higher MIC values (2.5 and 5.0 mg/mL, respectively). This was accompanied by a lack of apparent cytotoxicity to normal human BJ foreskin fibroblasts, enterocytes derived from CaCo2 cells, and zebrafish embryos. Further analyses revealed the presence of bioactive chlorogenic acids in the fractionated extract, especially in the ethyl acetate fraction (C). These findings support the traditional use of the root from C. acaulis and pave the way for the development of new formulations for treating bacterial infections. This was further evaluated in a proof-of-concept experiment where fraction C was used in the ointment formulation, which maintained high antimicrobial activity against MRSA and displayed low toxicity towards cultured fibroblasts.