Laba garlic is a traditional Chinese processed garlic (Allium sativum L.) with multiple health benefits. This study was aimed at isolating and identifying three antimicrobial peptide fractions from laba garlic using Sephadex chromatography, pre-HPLC and LC-MS/MS and investigating the potential antimicrobial mechanism. The sequences of the three peptides were demonstrated to be Tyr-Asn-His-Asn-Phe (YNHNF, F3-3-a), Trp-Pro-Thr-Ser-Phe-Thr (WPTSFT, F3-3-b) and Ala-Val-Asp-Arg-Ala-Val (AVDRAV, F3-3-c), respectively. F3-3-c showed the strongest anti-microbial effects among these three fractions and it could inhibit the mycelial growth of E. coli and S. aureus in vitro with a minimum inhibitory concentration (MIC) of 100 μM and exhibited negligible hemolytic activity. F3-3-c showed amphipathic properties with the presence of α-helix (25.8 ± 0.56%), β-strands (19.7 ± 0.45%), turns (21.2 ± 0.78%) and unordered conformation (33.3 ± 1.09%). Treatment of F3-3-c resulted in a change in the morphology of the hyphae and the disruption of membrane integrity as shown by SEM and TEM analyses. These results suggested that the laba garlic peptide F3-3-c might be a promising candidate for antibiotics and related functional foods.