Background: Chlorhexidine (CHX) is one of the most frequently used antiseptic agents in challenging multidrugresistant (MDR) isolates. There is an increasing number of reports on reduced susceptibility (tolerance) to CHX in MDR isolates. We aimed to investigate CHX tolerance in Gram-negative bacilli (GNB) and its possible association with antibiotic resistance. Methods: A total of 84 enteric GNB (ENT) and 40 non-fermentative GNB (NFGNB) isolates were collected from different clinical specimens. Disk diffusion method was performed to differentiate between MDR and non-MDR isolates and tolerance to CHX was determined by a modified agar dilution method. In GNB isolates, CHX tolerance was defined as a minimum inhibitory concentration (MIC) >= 4 mg/L. Results: We detected that 26.2% (22/84) of ENT and 50.0% (20/40) of NFGNB were MDR and the rest were nonMDR isolates. The CHX tolerance rate was detected as 50.0% (10/20) in MDR-NFGNB and 15.0% (3/20) in nonMDR-NFGNB, and this difference was statistically significant (p < 0.05). Conversely, the tolerance rate was observed as 4.5% (1/22) in MDR-ENT and 1.6% (1/62) in non-ENT, and this difference was not statistically significant (p > 0.05). Conclusions: NFGNB isolates had a higher tendency to CHX tolerance than ENT, and antibiotic resistance facilitates the selection of CHX tolerance in NFGNB but not in ENT isolates.