Abstract Background Chlorhexidine is a widely used disinfectant in clinical settings and a broad-spectrum antimicrobial agent effective against aerobic and anaerobic bacteria. However, disinfectant resistant or non-susceptible bacteria, including antibiotic-resistant Acinetobacter baumannii, have been found. This study aimed to develop a new technique to prevent and control A. baumannii infection in the hospital setting. Methods Chlorhexidine combined with minocycline, doxycycline, meropenem, imipenem, levofloxacin and ciprofloxacin were tested against the 30 multidrug-resistant and extremely drug-resistant A. baumannii clinical isolates. The checkerboard test was used to calculate the fractional inhibitory concentration index according to the minimum inhibitory concentration value for chlorhexidine combined with antibiotics. Results The combination of chlorhexidine with minocycline, doxycycline, meropenem, or ciprofloxacin showed synergistic responses in all clinical isolates, and more than 50% of isolates showed FICI ≤0.5. However, chlorhexidine together with imipenem or levofloxacin showed indifferent responses in 10% and 3.33% clinical isolates, respectively. In all tests, combinations of chlorhexidine with each of the above six antibiotics showed synergistic and additive effects, and inhibited the clinical isolates. Conclusions We concluded that, chlorhexidine combined with antibiotics could be used to control the risk of infection with A. baumannii.