Probiotics might be one of the solutions to reduce the effects of the recent ban on antimicrobial growth promoters in feed. However, the mode of action of probiotics still not fully understood. Therefore, evaluating probiotics (microbial feed additives) is essential. Thus the objective of this work was to investigate the efficacy of a new microbial feed additive (Lactobacillus salivarius and Lactobacillus reuteri) in broiler nutrition. The body weight (BW), average daily weight gain was relatively increased by the dietary inclusion of Lactobacillus sp. in broiler diets. Furthermore, the Lactobacillus feed additive influenced the histomorphological measurements of small intestinal villi. The addition of Lactobacillus sp. increased (p < 0.05) the villus height (VH)/crypt depth ratio and the VH was numerically increased in duodenum. The duodenal crypt depth remained unaffected (p > 0.05), while the ileal crypt depth was decreased by dietary supplementation of Lactobacillus sp. compared with the control. At the end of the feeding period, the basal and glucose stimulated short-circuit current (Isc) and electrical tissue conductivity were measured in the isolated gut mucosa to characterize the electrical properties of the gut. The addition of glucose on the mucosal side in Ussing chamber produced a significant increase (p = 0.001) in Isc in both jejunum and colon relative to the basal values in Lactobacillus probiotic group. This increase in Isc for probiotic group in jejunum is equivalent to an increase of about two times that for the basal values, while in the control group is about half fold that for the basal value. In addition, the DeltaIsc after glucose addition to the large intestine was greater than the DeltaIsc in the small intestine in both control and probiotic group. Moreover in both jejunum and colon, the increase in Isc for birds fed Lactobacillus was higher than their control counterparts (p < or = 0.1). This result suggests that the addition of Lactobacillus sp. to broiler diets increased the glucose transport. Additionally, the results indicated that the conductivity of jejunal and colonic tissues remained unaffected by the dietary inclusion of Lactobacillus and support the concept that this additive enhances the maintenance and function of the epithelial barrier. In conclusion, dietary inclusion of a microbial feed additive (L. salivarius and L. reuteri) slightly increased the growth performance and improved intestinal nutrient absorption which was in association with the intestinal architecture improvement.