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Environmental service workers may be exposed to pathogens during the cleaning of pathogen-containing bodily fluids.Participants with experience cleaning hospital environments were asked to clean simulated, fluorescein-containing vomitus using normal practices in a simulated patient room. Fluorescein was visualized in the environment and on participants under black lights. Fluorescein was quantitatively measured on the floor, in the air, and on gloves and shoe covers.In all 21 trials involving 7 participants, fluorescein was found on the floor after cleaning and on participants' gloves. Lower levels of floor contamination were associated with the use of towels to remove bulk fluid (ρ = −0.56, P = .01). Glove contamination was not associated with the number or frequency of contacts with environmental surfaces, suggesting contamination occurs with specific events, such as picking up contaminated towels. Fluorescein contamination on shoe covers was measured in 19 trials. Fluorescein was not observed on participants' facial personal protective equipment, if worn, or faces. Contamination on other body parts, primarily the legs, was observed in 8 trials. Fluorescein was infrequently quantified in the air.Using towels to remove bulk fluid prior to mopping is part of the recommended cleaning protocol and should be used to minimize residual contamination. Contamination on shoes and the floor may serve as reservoirs for pathogens.