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Objectives: Although routine disinfection of portable medical equipment is required in most hospitals, frontline staff may not be able to disinfect portable medical equipment at a rate that adequately maintains low bioburden on high-use equipment. This study quantified bioburden over an extended time period for two types of portable medical equipment, workstations on wheels and vitals machines, across three hospital wards. Methods: Bioburden was quantified via press plate samples taken from high touch surfaces on 10 workstations on wheels and 5 vitals machines on each of 3 medical surgical units. The samples were taken at three timepoints each day over a 4-week period, with random rotation of timepoints and portable medical equipment, such that frontline staff were not aware at which timepoint their portable medical equipment would be sampled. The mean bioburden from the different locations and portable medical equipment was estimated and compared with Bayesian multilevel negative binomial regression models. Results: Model estimated mean colony counts (95% credible interval) were 14.4 (7.7–26.7) for vitals machines and 29.2 (16.1–51.1) for workstations on wheels. For the workstations on wheel, colony counts were lower on the mouse, 0.22 (0.16–0.29), tray, 0.29 (0.22, 0.38), and keyboard, 0.43 (0.32–0.55), when compared to the arm, as assessed by incident rate ratios. Conclusions: Although routine disinfection is required, bioburden is still present across portable medical equipment on a variety of surfaces. The difference in bioburden levels among surfaces likely reflects differences in touch patterns for the different portable medical equipment and surfaces on the portable medical equipment. Although the association of portable medical equipment bioburden to healthcare-associated infection transmission was not assessed, this study provides evidence for the potential of portable medical equipment as a vector for healthcare-associated infection transmission despite hospital disinfection requirements.