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Two experiments were conducted to evaluate the in vitro and in vivo effects of a novel direct-fed microbial (DFM) containing <i>Lactobacillus animalis</i> LA-51, <i>Propionibacterium freudenreichii</i> PF-24, <i>Bacillus licheniformis</i> CH-200, and <i>Bacillus subtilis</i> King (BOVAMINE DEFEND^® Plus) against <i>Clostridium perfringens</i> pathogenic strains. In Experiment 1 (in vitro), an agar diffusion assay was performed to qualitatively evaluate the in vitro inhibitory effects of the DFM against <i>C. perfringens</i> types A and C. Including the DFM in the tested yielded inhibition zones with greater than three ring diameters in a 96-well plate. In Experiment 2 (in vivo), twenty 1-day-old beef calves were allocated to control (n = 10) or DFM (n = 10) for 21 days. All calves were orally challenged with 1.0 × 10⁸ colony forming units of <i>C. perfringens</i> type A strain S-107 per head. The procedures such as general health scores, body weight, and fecal sample collections were performed following the <i>C. perfringens</i> challenge. Daily feeding of DFM significantly reduced the incidence of diarrhea while improving general impression and appearance scores of calves. Overall, these results highlight the ability of the DFM containing <i>L. animalis</i> LA-51, <i>P. freudenreichii</i> PF-24, <i>B. licheniformis</i> CH-200, and <i>B. subtilis</i> (BOVAMINE DEFEND^® Plus) to inhibit <i>C. perfringens</i> types A and C under different experimental settings.