In many countries, traditional herbal medicines are gaining traction as potential alternative treatments for a variety of ailments. Medicinal plants have gained wider acceptance in recent years due to the belief that natural products are more effective and have fewer side effects than their synthetic counterparts. Glycyrrhiza glabra (G. glabra), commonly known as licorice root, a member of the Leguminoceae family, has been used as a medicinal plant in folk medicine since ancient times. A sugar-bearing plant, G. glabra contains five times more sugar than a typical plant. It contains many different bioactive sugars, including polysaccharides, flavonoids, terpenes, simple sugars, amino acids, and mineral salts. In research, we aimed to determine phenolic compounds by LC-MS/MS screening technique and to investigate its antimicrobial effect by MIC method using G. glabra methanol root extract. The inhibitory effect of G. glabra methanol extract on the growth of yeast (C. albicans) and pathogenic bacteria (E. coli, P. aeruginosa, S. aureus, and B. subtilis) was determined using the minimum inhibitory concentrations (MIC) method. A total of twenty-two compounds were identified as a result of the study. The main compounds detected in the sample were cynarin (11.62 µg g⁻¹), chlorogenic acid (8.75 µg g⁻¹), and p-coumaric acid (3.88 µgg⁻¹). The extract was determined to exhibit highly effective inhibition against C. albicans yeast, effective inhibition against P. aeruginosa, and mild inhibition against other bacteria. The findings showed that licorice root may be a natural alternative to drugs because it contains a large number of biochemicals, is selectively toxic, and inhibits the growth of some pathogenic bacteria.