A lactic acid bacterium showing antimicrobial activity against fish pathogen was isolated from gastrointestinal tract of flounder for the purpose of use as an aquaculture probiotics. From the analysis of morphological and physiological characteristics, the isolated strain was named as Lactococcus sp. HM58. Antimicrobial substance (AMS) from Lactococcus sp. HM58 showed strong growth inhibitory activity against Streptococcus sp., which is a fish pathogenic bacterium. AMS was presumed a proteinaceous compound with stability in heat and wide pH range from 2 to 10. It was started to produce in exponential growth phase and was not produced any more in stationary phase. It showed comparatively broad antimicrobial spectrum against most of gram positive bacteria used for this study. About $84\%$ of Lactococcus sp. HM58 was able to survive in the artificial gastric juice though it was low to the extent in the artificial bile juice. In the sensitivity test for various antibiotics, this strain was highly sensitive for doxycycline, erythromycin, amoxicillin clavu1anic acid and ampicillin.