BackgroundsAntiseptic bathing did not reduce central-line (CL) associated bloodstream infection (CLABSI) rates in intensive care units (ICU) according to a recent cluster randomised controlled trial (cRCT). However, this analysis did not consider baseline infection rates. Our post-hoc analysis of this cRCT aimed to use a before-after comparison to examine the effect of daily bathing with chlorhexidine, octenidine or water and soap (control) on ICU-attributable CLABSI rates.MethodsA post-hoc analysis of a multi-center cRCT was done. ICUs that did not yet perform routine antiseptic bathing were randomly assigned to one of three study groups applying daily bathing with 2% chlorhexidine-impregnated cloths, 0.08% octenidine wash mitts or water and soap (control) for 12 months. Baseline data was assessed 12 months before the intervention started when all ICUs routinely used water and soap. Poisson regression and generalised estimating equation models were applied to identify changes of CLABSI rates per 1000 CL days between intervention and baseline periods in each study group.ResultsThe cRCT was conducted in 72 ICUs (24 per study group) including 76,139 patients in the baseline and 76,815 patients in the intervention period. In the chlorhexidine group, incidence density of CLABSI was reduced from 1.48 to 0.90 CLABSI per 1000 CL days comparing baseline versus intervention period (P= 0.0085). No reduction was observed in the octenidine group (1.26 versus 1.47 CLABSI per 1000 CL days,P= 0.8735) and the control group (1.20 versus 1.17,P= 0.3298). Adjusted incidence rate ratios (intervention versus baseline) were 0.63 (95%CI 0.46–0.87,P= 0.0172) in the chlorhexidine, 1.17 (95% CI 0.79–1.72,P= 0.5111) in the octenidine and 0.98 (95% CI 0.60–1.58,P= 0.9190) in the control group. Chlorhexidine bathing reduced CLABSI with gram-positive bacteria, mainly coagulase-negative staphylococci (CoNS).ConclusionsIn this post-hoc analysis of a cRCT, the application of 2% chlorhexidine-impregnated cloths reduced ICU-attributable CLABSI. This preventive effect of chlorhexidine was restricted to CLABSI caused by gram-positive pathogens (CoNS). In contrast, 0.08% octenidine wash mitts did not reduce CLABSI rates in ICUs.Trial registrationRegistration number DRKS00010475, registration date August 18, 2016.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13756-023-01260-w.