Direct-fed microorganisms (DFM) and exogenous enzymes have been demonstrated to improve growth performance in poultry and are potentially important alternatives to antibiotic growth promoters (AGP). We investigated the administration of a feed additive composed of a DFM product containing spores of 3 Bacillus amyloliquefaciens strains and an enzyme blend of endo-xylanase, α-amylase, and serine-protease in diets with or without sub-therapeutic antibiotics in broiler chickens over a 42-d growth period. Evaluation of growth performance determined feed efficiency of broiler chickens which were administered the feed additive was comparable to those fed a diet containing AGPs. Characterization of the gastrointestinal microbiota using culture-dependent methods determined administration of the feed additive increased counts of total Lactic Acid Bacteria (LAB) relative to a negative control and reduced Clostridium perfringens to levels similar to antibiotic administration. Additionally, greater counts of total LAB were observed to be significantly associated with reduced feed conversion ratio, whereas greater counts of C. perfringens were observed to be significantly associated with increased feed conversion ratio. Our results suggest the co-administration of DFMs and exogenous enzymes may be an important component of antibiotic free poultry production programs and LAB and C. perfringens may be important targets in the development of alternatives to AGPs in poultry production.