Gelatinase is an extracellular metalloprotease and is capable of hydrolyzing gelatine, collagen, elastin, etc., which is used in processing industries, food technology and research. In this study, 216 bacterial strains isolated from diseased fishes were examined their ability to produce gelatinase. As a result, eleven strains (5.09%) were positive for gelatinase production. Gelatinase activity ranged from 0.3 to 0.64 U/ mL, in which the strain MD4 showed the highest gelatinase activity (0.64 ± 0.11 U/mL). Strain MD4 grew in the range of temperature from 25 to 45°C (optimum at 37°C), pH 4.0 ÷ 10.0 (optimum at pH 7.0), and NaCl concentration from 0.5 to 5% (optimum at 4%). Strain MD4 was characterized as Gram-positive, spheroidal, non-spore-forming, non-spore organism. As a consequence, strain MD4 was selected and genetically identificated using 16S rRNA gene sequence analysis. The 16S rRNA sequence of strain Enterococus faecalis MD4 (GenBank accession No. MG982575.1.) shared 99% identity with Enterococus faecalis NBRC 100480.