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ABSTRACT Prenatal screening in pregnant women between 35 and 37 weeks of gestation and intrapartum antibiotic prophylaxis has successfully reduced the incidence of neonatal morbidity and mortality related to Streptococcus agalactiae. However, the contamination rates of newborns are still considerable. In traditional and folk medicines, it has been observed that garlic has been effective in treating S. agalactiae infection. The aim of this study was to isolate and identify the active compounds from garlic that have antimicrobial activity against S. agalactiae. In order to do this, SP80 (Sep-Pak 80%) obtained from crude garlic extract (CGE) was fractionated by reverse-phase ultrafast liquid chromatography with UV (RP-UFLC-UV) using a Shim-pack PREP-ODS column. All fractions obtained were tested using a microbial growth inhibition test against the S. agalactiae strain (ATCC 12386). Five clinical isolates were used to confirm the action of the fractions with antimicrobial activity, and the bacterial growth curve was determined. Identification of the antimicrobial compounds was carried out through liquid chromatography coupled with mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). The active compounds found to exhibit antimicrobial activity were Ƴ-glutamyl-S-allyl-cysteine (fraction 18), Ƴ-glutamyl-phenylalanine (fraction 20), and the two stereoisomers (E and Z) of ajoene (fraction 42). The MICs of these fractions were 5.41 mg/ml, 4.60 mg/ml, and 0.16 mg/ml, respectively, and they inhibited the growth of the clinical isolates tested. Antimicrobial compounds from garlic may be a promising source in the search for new drugs against S. agalactiae. IMPORTANCE Invasive disease due to group B streptococcal (GBS) infection results in a wide spectrum of clinical disease in neonates. Maternal colonization by GBS is the primary risk factor for disease. The strategy recommended by the Centers for Disease Control to reduce neonatal GBS infection is the culture-based screening of all pregnant women at 35 to 37 weeks of gestation and intrapartum antibiotic prophylaxis (IAP). However, indiscriminate use of antibiotics favors the selection and spread of resistant bacteria. The global scenario of antibacterial resistance has been of great concern for public health, and natural products can be a source of new substances to help us grapple with this problem.