The present study was conducted to select appropriate microbial strains for the development of fermented Alisma canaliculatum A. Br. et Bouche (FAC) feed additive and to examine their effects on broilers. In experiment 1, 16 strains of Lactobacillus acidophilus, Lactobacillus plantarum, Enterococcus faecium, Bacillus subtilis, Bacillus coagulans, and Saccharomyces cerevisiae from the Korean Collection for Type Cultures (KCTC) were evaluated. The strains were tested for their acid, bile, and heat tolerance levels. Among them, L. acidophilus KCTC 3111, E. faecium KCTC 2022, B. subtilis KCTC 3239, and S. cerevisiae KCTC 7928 were selected to produce FAC. For experiment 2, 140 Ross broiler chicks were allocated to four 5-wk-long dietary groups: NC (basal diet), PC (basal diet with 0.005% oxytetracycline), AC (basal diet with 0.5% A. canaliculatum rhizome powder), and FAC (basal diet with 0.5% FAC). Final BW and total BW gain were increased (P < 0.05) upon the addition of FAC, whereas feed conversion ratio improved (P < 0.05) in the supplemented groups compared with the NC group. Crude protein content increased and crude fat decreased (P < 0.05) in the breast meat of the FAC group compared with the AC and PC groups, respectively. Proventriculus and kidney relative weights of the FAC group differed (P < 0.05) from the AC group but not from the PC group. Lower (P < 0.05) thiobarbituric acid reactive substances value of thigh meat was observed in the FAC group compared with the other groups. The FAC diet increased (P < 0.05) α-linolenic acid and linoleic acid in meat. Alteration of these fatty acids led to increased levels of polyunsaturated fatty acid, polyunsaturated fatty acid /saturated fatty acid, n-3 and n-6 fatty acids, and decreased n-6/n-3 ratio in breast or thigh meat (P < 0.05). It is concluded that FAC confers high tolerance to acid, bile and heat; and beneficially affects growth, meat composition, oxidative stability, and fatty acid composition. The FAC may be a functional feed additive in broilers.