Calves are often suffering from diarrhea. The previous researches showed that addition of two selected strains, Lactobacillus plantarum Chikuso-1 and Candida sp. CO119 to milk replacer significantly suppressed calf diarrhea and increased the number of fecal Lactic Acid Bacteria (LAB). It remains unknown however, whether feeding of the microbe could affect bacterial flora in the specific areas of gastrointestinal tract in host animals. In the present study, the effects of feeding of Chikuso-1 and CO119 (microbe-fed) on bacterial flora of various gastrointestinal regions were examined in Holstein calves. Gastrointestinal contents of control and microbe-fed calves were collected from rumen, duodenum, jejunum, ileum, cecum and colon, respectively and immediately inoculated on the selective agar media for LAB, coliform, aerobic bacteria, bacilli and clostridia for bacterial enumeration. Feeding of the microbe significantly increased the number of LAB and LAB/coliform ratio in rumen, duodenum and jejunum. The present results suggest that feeding of Chikuso-1 and CO119 improves bacterial flora on rumen and upper small intestine in the gastrointestinal tract of Holstein calves.