Background Annually, almost 2 billion peripheral intravenous cannulas (PIVCs) are placed worldwide. In response to concerns of infectious complications, chlorhexidine is increasingly utilised for maintenance decontamination of PIVC injection ports. Concomitantly, the allergenic potential of chlorhexidine exposures has been highlighted by several case reports, implicating it as a trigger when used for this seemingly innocuous process. Given how widespread this application is, elucidating potential chlorhexidine exposure is needed to gauge its risks and benefits. Objective To examine and quantify if chlorhexidine is entrained when used for PIVC injection port cleaning. Methods Twenty benchtop PIVC set‐ups were cleaned with 2% chlorhexidine and 70% alcohol wipes, following three different pragmatic protocols. Each set‐up was injected with 10 ml ultrapure water, and samples tested by liquid chromatography‐electrospray tandem mass spectrometry for entrained chlorhexidine. Results Chlorhexidine was detected in every sample. Mean concentrations and standard deviations from each protocol were 41.47 ppb (4.08), 54.76 ppb (17.46), and 65.84 ppb (7.01). One‐way ANOVA indicated a statistical difference between at least two groups (df = 2, F = 24.11, p < .00001), with Tukey's testing verifying significantly different mean concentrations between all groups (p < .01). Conclusions Using 2% chlorhexidine and 70% alcohol swabs to decontaminate PIVC injection ports resulted in consistent entrainment of chlorhexidine, with varying amounts correlated to how it was applied. These results validate case reports attributing anaphylactic/allergic reactions to suspected intravenous chlorhexidine entrainment and should factor into future risk–benefit assessments for its use in PIVC maintenance antisepsis policies and protocols.