Vibrio sp. is the most serious pathogen in marine aquaculture, and the development of anti-Vibrio agents is urgently needed, but there is an extreme lack of high-throughput screening (HTS) models for searching anti-Vibrio compounds. Here, we established a protein-based HTS screening model to identify agents targeting peptide deformylase (PDF) of Vibrio anguillarum. Crude extracts derived from marine actinomycetes were applied for screening with this model, and the crude extract of strain Streptomyces sp. NHF165 dramatically inhibited both V. anguillarum PDF (VaPDF) activity and V. anguillarum cell growth. Actinonin was further identified as the functional component. The anti-VaPDF and anti-V. anguillarum activities of actinonin were dose-dependent, with IC50 values of 6.94 and 2.85 µM, respectively. To understand the resistance of V. anguillarum against actinonin, spontaneous resistant mutants were isolated, and a deletion (774-852 base pairs) was found in the folD gene (which produces 10-formyl-tetrahydrofolate, a donor of N-formyl to Met-tRNAfmet) in resistant strains. Compared to the wild type strain, the folD mutant showed eight times the minimum inhibition concentration on actinonin, while the folD complementary strain could not grow on medium supplemented with actinonin, suggesting that folD gene mutation was mainly responsible for actinonin resistance. To our knowledge, this is the first report showing that marine-derived Streptomyces sp. could produce actinonin with anti-VaPDF activity and that the resistance of V. anguillarum against actinonin is mediated by mutation in the folD gene.