ObjectiveProbiotics have been used in poultry production to improve the performance and health of chickens raised without antibiotics. The combination of different probiotic strains has been used with the hope of conferring multiple benefits to the host. However, the inclusion of several strains does not necessarily boost benefits. There is a lack of studies that compare the efficacy of multi-strain probiotics to their individual components. In this study, the effects of aBacillus-based probiotic product mix containingB. coagulans,B. licheniformis,B. pumilus, andB. subtilisagainstClostridium perfringenswere tested in vitro using a co-culture method. The individual strains and different combinations of the strains used in the product were also tested againstC. perfringens.ResultsThe probiotic product mix tested in this study did not show effects againstC. perfringens(P= 0.499). When tested individually, the strain ofB. subtiliswas the most efficient strain to decreaseC. perfringensconcentrations (P≤ 0.01), and the addition of otherBacillusspecies strains significantly decreased its efficacy againstC. perfringens. We concluded that the probiotic mix ofBacillusstrains used in this study (B. coagulans,B. licheniformis,B. pumilusandB subtilis) was not effective in decreasingC. perfringensconcentrations in vitro. However, when deconstructing the probiotic, the strain ofB. subtilisalone or combined with the strain ofB. licheniformiswere effective againstC. perfringens. This suggests that the anticlostridial properties of the particular strains ofBacillusused in this study were negatively affected when combined with otherBacillusspp. strains.