Acinetobacter baumannii is the major nosocomial pathogen that causes serious infections such as ventilator-associated pneumonia and bacteremia due to its biofilms. Hence, this study investigated the antimicrobial and antibiofilm potentials of cell-free supernatants (CFS) obtained from Clostridium butyricum, as probiotic, against A. baumannii. Our results demonstrated that C. butyricum CFS inhibited A. baumannii cell growth in planktonic culture. Also, C. butyricum CFS not only inhibited the biofilm development and dispersed mature biofilms, but also suppressed the metabolic activity of biofilm cells, showing antibiofilm activity. The biofilm components reduced by C. butyricum CFS were observed via confocal laser scanning microscopy. In addition, C. butyricum CFS exhibited antivirulence effect by inhibiting the motility of A. baumannii. Furthermore, C. butyricum CFS significantly downregulated the expression of efflux pump-related genes including adeA, adeB and adeC in A. baumannii. Our data demonstrate that C. butyricum CFS showed antimicrobial and antibiofilm effects on A. baumannii. These effects are closely associated with suppression of motility and efflux pump-related genes in A. baumannii. The findings suggest that C. butyricum CFS can be used as a new therapeutic alternative against biofilm-associated infection caused by multidrug-resistant A. baumannii.