() is an important Gram-positive food-borne pathogen that severely threatens public health. A checkerboard microdilution method was performed to evaluate the synergistic effect of lithocholic acid (LCA) with Gentamicin (Genta) against . BacLight LIVE/DEAD staining, scanning electron microscopy and biofilm inhibition assays were further used to explore the bactericidal effect and antibiofilm effect of this combination on . Additionally, the synergistic effects of LCA derivatives with Genta were also evaluated against , and The results indicated that a synergistic bactericidal effect was observed for the combined therapy of LCA at the concentration without affecting bacteria viability, with Genta. Additionally, LCA in combination with Genta had a synergistic effect against Gram-positive bacteria (, and ) but not against Gram-negative bacteria (, and ). BacLight LIVE/DEAD staining and scanning electron microscopy analysis revealed that the combination of LCA with Genta caused membrane injury, leading to bacteria death. We found that 8 μg/mL LCA treatment effectively improved the ability of Genta to eradicate biofilms. In addition, we found that chenodeoxycholic acid, as a cholic acid derivative, also improved the bactericidal effect of Genta against Gram-positive bacteria. Our results indicate that LCA represents a broad-spectrum adjuvant with Genta for infection caused by and other Gram-positive pathogens.