The barramundi (Lates calcarifer) and fourfinger threadfin (Eleutheronema tetradactylum) are economically important farmed fishes in Taiwan. Due to intensive farming and extreme weather conditions, they are prone to bacterial diseases, especially streptococcal infection. Lonicera japonica is rich in polyphenolic compounds and has broad antibacterial activity. In order to enhance disease resistance of fish, this study aimed to prepare extracts of L. japonica to examine their antibacterial ability in vitro and their effects on resistance to Streptococcus iniae infection of the barramundi and fourfinger threadfin. The results showed that the total phenolic content of L. japonica extract was 2.4 times higher than that of the crude drug source, and its chlorogenic acid content was 7%. An extract concentration less than 281 μg/ml had no effect on catfish gill cell proliferation. The barramundi and fourfinger threadfin were fed diets containing 0, 1, 2, 3, and 4% L. japonica extract for 14 and 28 days, respectively, and then challenged with S. iniae. The results showed that disease resistance of all groups given L. japonica extract was significantly better than that of the control group (p < 0.01), and the highest protection afforded was more than 50%. In conclusion, the best application method of L. japonica extract is 3% in the feed of barramundi for 14 days and 4% in the feed of fourfinger threadfin for 28 days.