In this study, we examined DNA sequences of bacteria isolated from goldfish intestine and identified the species in collaboration with the Food Industry Research & Development Institute of Taiwan. One bacterial species, Enterococcus avium, was selected for evaluation of its antimicrobial effects and probiotic function. The antimicrobial effects of feed containing E. avium were investigated in tilapia challenged with Streptococcus agalactiae. We found that E. avium inhibits a variety of pathogens, including gram-positive and gram-negative bacteria, with the strongest inhibition observed for Pseudomonas aeruginosa. Moreover, E. avium cells disrupted by sonication retained their antibacterial effects. By treating the sonicated cells at different temperatures or pH, we discovered that heating the cells above 75 °C resulted in the loss of their inhibitory effects. Moreover, E. avium pellets resuspended at pH 3.0 exhibited strong bacteriostatic activity, whereas E. avium pellets resuspended in solutions of pH > 10 had diminished antimicrobial activity. Tilapia fed once or twice a day with high concentrations (1 × 10¹⁰, 1 × 10⁹, or 1 × 10⁸ cfu/g) of sonicated or viable bacteria exhibited higher growth rates than both the control group and the experimental group fed 1 × 10⁷ cfu/g. In addition, tilapia fed high concentrations of sonicated bacteria twice a day exhibited a higher survival rate than fish fed viable bacteria after a challenge with S. agalactiae.