Background: A number of studies have shown that daily bathing by chlorhexidine (CHG) wipes in ICU can reduce healthcare-associated infections (HAIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). However, the impact of CHG bathing on carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Enterobacteriaceae (CRE) is not clear and remains controversial. Methods: A semi-experimental study which employs both pre-controls and a parallel control was conducted. In the intervention period (from July 1 to December 31, 2016), strengthened infection control measures and daily bathing with 2% CHG-impregnated wipes once daily was performed in the ICU. Fifty-seven non-ICU wards with the occurrence of multidrug-resistant organisms (MDRO) infections during the same time period were selected as parallel control group (only CHG bathing was not performed). The net effect of the 2% CHG daily bathing was evaluated by the difference in difference (DID) model.Results: The incidence of HAIs caused by CRPA in ICU was significantly decreased between the intervention and pre-intervention period (2.5, 95% confidence interval (CI) 1.6–3.8 vs. 4.6, 95% CI 3.3–6.3 cases /1,000 patient days, P = 0.02). Similarly, the incidence of HAIs caused by CRAB in intervention group was 19.75% lower than that in pre-intervention group (6.0, 95% CI 4.4–7.6 vs. 7.5, 95% CI 5.7–9.3 cases / 1,000 patient days, P = 0.24). The DID model analysis showed that CHG bathing reduced the incidence of CRAB- and CRPA-caused infections in ICU by 1.56 and 2.15 cases/1,000 patient days, respectively, P <0.01, and bathing of every 38 patients (95% CI, 21–268) and 39 patients (95% CI, 24–110) were able to prevent one case of HAIs of CRAB and CRPA, respectively. However, CHG bathing showed no effect on MRSA, VRE and CRE (P>0.05).Conclusion: Daily bathing with 2% CHG-impregnated wipes can reduce HAIs caused by CRAB and CRPA, while it is not effective for the prevalence of infections caused by MRSA, VRE, and CRE.