<p><i>Mitsuokella multacida</i> AGMB01124 isolated from the feces of healthy swine was evaluated for its safety and potential as a next-generation probiotic (NGP). These bacteria have been increasingly reported to be positively correlated with short-chain fatty acids (SCFA), which are a beneficial energy source in the gut environment. We investigated the potential application of <i>M</i>. <i>multacida</i> AGMB01124 as an NGP by examining its genomic features, probiotic properties, cytotoxicity, oral toxicity, and its effect on SCFA levels in feces. Genome analysis did not identify any virulence-associated factors, and the strain harbored two antibiotic resistance genes (<i>tetW/N/W</i> and <i>ermB</i>). Furthermore, endoglucanase D (CelD) exhibited differences in four surface-exposed amino acid residues, three of which were located near the active site and one within the signal peptide region, suggesting a role in modulating catalytic function. Phenotypically, the strain exhibited susceptibility to most antibiotics recommended by Clinical &amp; Laboratory Standards Institute and was non-hemolytic, non-cytotoxic. The strain demonstrated some tolerance to acidic and bile conditions, strong self-aggregation, co-aggregation with pathogens, and low cell surface hydrophilicity. Oral toxicity studies indicated no adverse effects on clinical signs, body weight, diet intake, relative organ weights, hematology, serum biochemistry, histopathology, or harmful enzyme activities. Short-chain fatty acid concentrations were significantly higher in the <i>M</i>. <i>multacida</i> AGMB01124-administered group than in the control group, suggesting potential contributions to host gut health and metabolic regulation. Therefore, this study provides preliminary evidence supporting the safety of the <i>M</i>. <i>multacida</i> AGMB01124 strain and its potential application as a candidate NGP.</p>