OBJECTIVETo determine the typical microbial bioburden (overall bacterial and multidrug-resistant organisms [MDROs]) on high-touch healthcare environmental surfaces after routine or terminal cleaning.DESIGNProspective 2.5-year microbiological survey of large surface areas (>1,000 cm2).SETTINGMDRO contact-precaution rooms from 9 acute-care hospitals and 2 long-term care facilities in 4 states.PARTICIPANTSSamples from 166 rooms (113 routine cleaned and 53 terminal cleaned rooms).METHODSUsing a standard sponge-wipe sampling protocol, 2 composite samples were collected from each room; a third sample was collected from eachClostridium difficileroom. Composite 1 included the TV remote, telephone, call button, and bed rails. Composite 2 included the room door handle, IV pole, and overbed table. Composite 3 included toileting surfaces. Total bacteria and MDROs (ie, methicillin-resistantStaphylococcus aureus, vancomycin-resistant enterococci [VRE],Acinetobacter baumannii,Klebsiella pneumoniae, andC. difficile) were quantified, confirmed, and tested for drug resistance.RESULTSThe mean microbial bioburden and range from routine cleaned room composites were higher (2,700 colony-forming units [CFU]/100 cm2; ≤1–130,000 CFU/100 cm2) than from terminal cleaned room composites (353 CFU/100 cm2; ≤1–4,300 CFU/100 cm2). MDROs were recovered from 34% of routine cleaned room composites (range ≤1–13,000 CFU/100 cm2) and 17% of terminal cleaned room composites (≤1–524 CFU/100 cm2). MDROs were recovered from 40% of rooms; VRE was the most common (19%).CONCLUSIONSThis multicenter bioburden summary provides a first step to determining microbial bioburden on healthcare surfaces, which may help provide a basis for developing standards to evaluate cleaning and disinfection as well as a framework for studies using an evidentiary hierarchy for environmental infection control.Infect Control Hosp Epidemiol2016;1426–1432