The escalating crisis of antimicrobial resistance in aquaculture, driven by the indiscriminate use of antibiotics, underscores the urgent need to develop novel anti-infective agents. This study addresses this requirement by investigating cysteine-rich antimicrobial peptides (AMPs) in understudied crustacean species. A cysteine-rich AMP, designated PpRcys1, was identified and characterized from the genome of Pollicipes pollicipes. PpRcys1 comprises 104 amino acids, with 85 residues forming the mature peptide region, and exhibits random coils, a CSαβ-fold, and one β-sheet. Our findings demonstrated that recombinant PpRcys1 (rPpRcys1) possesses broad-spectrum antimicrobial activity against three Gram-positive bacteria (Staphylococcus aureus, Bacillus sp. T2, and Streptococcus agalactiae) and four Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, Vibrio alginolyticus, and Acinetobacter sp. L3), with minimum inhibitory concentrations ranging from 8 to 32 μM. It exerts antimicrobial effects by inducing membrane disruption without impacting bacterial protease activity, DNA migration, or respiratory chain reductase activity. Further investigation is warranted to determine whether it can target and interfere with intracellular bacterial processes. Our discovery and characterization of this novel AMP provide a promising foundation for its development as an alternative to antibiotics.