Background and Aim:The use of antibiotics is associated with many side effects, with the development of bacterial resistance being particularly important. It has been found that dogs and their owners host similar resistant bacteria. This contributes to increased concurrent bacterial resistance and a possible trend of increased bacterial resistance in humans. Thus, using probiotics in dogs is an alternative option for preventing and reducing the transmission of bacterial resistance from dogs to humans. Probiotics are characterized by their potential to endure low pH levels and high concentrations of bile acids in the gastrointestinal tract. Lactobacilli are more acid-tolerant and resistant to bile acid, so they are ideal probiotics to be added to the canine diet. According to the previous studies, the benefits ofLactobacillusare a stable nutritional status and greater digestibility, along with improved fecal scores and reduced ammonia in dogs. However, no studies have been conducted withLactobacillus plantarumCM20-8 (TISTR 2676),Lactobacillus acidophilusIm10 (TISTR 2734),Lactobacillus rhamnosusL12-2 (TISTR 2716),Lactobacillus paracaseiKT-5 (TISTR 2688), andLactobacillus fermentumCM14-8 (TISTR 2720), or their use in combination. Hence, the aim of this study was to examine the possible effects of the aforementionedLactobacilluson hematological indices, nutritional status, digestibility, enzyme activities, and immunity in dogs. From the results, a new and safe strain ofLactobacillusmay emerge for use as a probiotic in the future.Materials and Methods:In this study, 35 dogs were allocated equally into seven groups: Group 1 received a basal diet (control), while Groups 2–7 received the same diet further supplemented withL. plantarumCM20-8 (TISTR 2676),L. acidophilusIm10 (TISTR 2734),L. rhamnosusL12-2 (TISTR 2716),L. paracaseiKT-5 (TISTR 2688),L. fermentumCM14-8 (TISTR 2720), or a mixture of probiotics (L. plantarum,L. acidophilus,L. rhamnosus,L. paracasei, andL. fermentum), respectively. All probiotics were administered at a dose of 109colony-forming unit/dog for 28 days. Nutritional status, hematology, serum biochemistry, digestibility, enzyme activities, and immunity parameters were assessed.Results:There were no differences among the groups in body weight, feed intake, body condition score, fecal score, and fecal dry matter on the different sampling days. The hematology and serum biochemical analyses showed a difference only in creatinine activity (p < 0.001), with higher values in groupL. fermentumCM14-8 (TISTR 2720) and lower values in groupL. paracaseiKT-5 (TISTR 2688) than in controls. However, all measurements were within the normal laboratory reference ranges. Fecal characteristics (fecal ammonia and fecal pH), fecal digestive enzyme activities, serum immunoglobulin (IgG), and fecal IgA did not differ significantly among the groups (p > 0.05).Conclusion:Lactobacillus plantarumCM20-8 (TISTR 2676),L. acidophilusIm10 (TISTR 2734),L. rhamnosusL12-2 (TISTR 2716),L. paracaseiKT-5 (TISTR 2688), andL. fermentumCM14-8 (TISTR 2720), along with their mixture are safe and non-pathogenic additives for use as new probiotic strains ofLactobacillusin dogs. Although the newLactobacillusstrains had no effect on hematology, serum biochemistry, nutritional status, digestive enzyme activities, immunity, body weight, feed intake, or body condition scores in dogs, further studies should investigate the intestinal microbiota and the development of clinical treatments.