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(1) Background: Based on the hazard of Streptococcus agalactiae to human and animal health and the increasing drug resistance, it is urgent to develop new antimicrobial agents with high bactericidal activity and low drug resistance against S. agalactiae. This study aims to investigate in vitro pharmacodynamics and bactericidal mechanism of fungal defensin-derived peptides NZX and P2 against S. agalactiae. (2) Methods: Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined by broth dilution method and AGAR plate dilution method. Cell membrane integrity was determined by flow cytometer. Cell morphological changes were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). (3) Results: MIC values (NZX: 0.11 μM, P2: 0.91 μM) and MPC (NZX: 1.82 μM) showed their higher antibacterial activity and stronger inhibition ability of drug resistance mutation. The bactericidal mechanism was elucidated that P2 caused S. agalactiae ACCC 61733 cells to deform, bound to the cell wall, and perturbed cell membrane, resulting in K+ leakage, membrane hyperpolarization, ATP release, and reduced cell contents. Compared with P2, NZX focuses on the cell wall, and it bound to the cell wall causing cells boundary disappearance. (4) Conclusion: NZX and P2 are promising antimicrobial agents for streptococcicosis treatment. [ABSTRACT FROM AUTHOR]