Effects of turkey-derived beneficial bacteria Lactobacillus ingluviei UMNPBX19 and Lactobacillus salivarius UMNPBX2 on Salmonella Heidelberg (SH) in turkey poults was investigated. Using in vitro studies, we determined each strain's resistance to pH 2.5 and 0.3% bile salts and their β-hemolysis activity. We also tested each strain's adherence to avian epithelial cells and exhibition of antimicrobial activity against major poultry-associated Salmonella. Moreover, using three in vivo experiments, we determined the effect of the strains in combination (LBIS) against SH in turkey poults. The treatment groups were negative control (−SH, −LBIS), SH control (+SH, −LBIS), and LBIS group (+SH, +LBIS). Supplementation of LBIS was done in drinking water throughout the study at a dose of 8 log CFU/gal. On day 7, poults were challenged with a 2011 ground turkey outbreak strain of SH at 5 × 105 CFU/mL, and the surviving pathogens were determined on day 7 postinoculation from the cecum, spleen, and liver. Both Lactobacillus strains exerted resistance to low pH and bile salts (P < 0.05), showed adhesion to epithelial cells (P < 0.05), but did not exhibit β-hemolysis. Cell-free culture supernatants of strains showed antimicrobial activity against Salmonella (P < 0.05). Results from the in vivo studies revealed that LBIS significantly reduced dissemination of SH to the liver and spleen in all experiments, and colonization in the cecum in two of the three experiments (1.9- and 3.9-log CFU/g reductions), compared with the control. The results indicate that turkey-derived L. ingluviei UMNPBX19 and L. salivarius UMNPBX2 have potential beneficial effects against SH in turkeys. However, more studies to this effect are warranted.