Abstract Probiotics including Lactobacillus and Bifidobacterium spp. have been known to control the balance of gut microbiota and are positively effective to host animals. In this study, 32 bacterial strains were isolated from Norwegian forest cat (castrated, 12 years old) and Persian cat (castrated, 10 years old) which were privately owned and had indoor access. Among them, Lactobacillus rhamnosus CACC612 (CACC612) and Bifidobacterium animalis subsp. Lactis CACC789 (CACC789) were selected as potential probiotics. From the characterization of the two strains, they showed equivalent acid tolerance, similar cell adhesion rate on HT-29 monolayer cell line, and superior bile salt tolerance, compared with Lactobacillus rhamnosus GG (LGG). Subsequently, they exhibited antibacterial activities against a broad spectrum of pathogenic bacteria such as E. coli KCTC 2617, Salmonella Derby NCCP 12238, Salmonella enteritidis NCCP 14546, Salmonella typhimurium NCCP 10328, Clostridium difficile JCM 1296T. From evaluating host effects, the viability of the feline macrophage cell line (Fcwf-4) increased with the treatment of CACC612 or CACC789 (P < 0.05). The induced expression of immune-related genes such as IFN-γ, IL1β, IL2, IL4, and TNF-α by immune stimulation was significantly attenuated by the treatment of CACC612 or CACC789 (P < 0.05). In addition, when 52 clinical factors of sera from 21 healthy cats were analyzed using partial least-squares discriminant analysis (PLS-DA), the animals were markedly clustered before and after CACC612 or CACC789 feeding. In addition, hemoglobin and Mean corpuscular hemoglobin concentration (MCHC) were significantly increased after CACC612 feeding (P < 0.05). Conclusively, these results could contribute to understanding the functional effects of feline-originated probiotics and supporting their industrial applications.