Along with the wide spread of bacterial antibiotic resistance over the world, the treatment efficiency of infectious disease is greatly affected by the mixed biofilm formation by pathogenic bacteria. Staphylococcus aureus and Pseudomonas aeruginosa, a frequent cause of nosocomial infections, exhibit both synergistic and antagonistic interactions in co-culture, leading to various changes in the metabolic profile of bacteria, which in turn affect their sensitivity to antimicrobials. Here we show that S. aureus cell-free culture liquid exhibits bacteriostatic properties and increases the efficacy of antimicrobials against P. aeruginosa. Thus, the MICs of amikacin, gentamicin, and ciprofloxacin decreased 2-4 fold in the presence of cell-free supernatant of S. aureus 24 h culture. Furthermore, the combination of the latter with antimicrobials increased the efficacy of amikacin up to 64-fold. Thus, the combined use of cell-free culture liquid of S. aureus with broad-spectrum antibiotics can be used to increase the effectiveness of antimicrobial therapy of P. aeruginosa.