Background: Occupational contact dermatitis is common in healthcare workers. Although irritant contact dermatitis resulting from wet work is the most frequently reported cause, healthcare workers also constitute high-risk group for the development of allergic contact dermatitis and contact urticaria. Objectives: To evaluate the prevalence of delayed-type and immediate-type hypersensitivity in 120 healthcare workers with hand eczema. Methods: One hundred and twenty healthcare workers from three major hospitals in Denmark with self-reported hand eczema within the last year participated in the study. Patch tests included baseline series plus selected allergens, and prick tests included standard inhalational allergens plus natural rubber latex and chlorhexidine. Levels of IgE specific for latex, chlorhexidine and ethylene oxide were measured. Results: Of the participants, 53% had positive patch test reactions. The most frequent positive patch test reactions were to nickel, thiomersal, fragrances, rubber chemicals, and colophonium. The prevalence of natural rubber latex allergy as diagnosed by prick testing was 2.5%, and chlorhexidine allergy (both contact allergy and IgE-mediated allergy) was found in <1%. Ethylene oxide allergy was not identified in any of the participants. Conclusions: Our results confirm previous reports on contact allergy patterns in healthcare workers. Testing for natural rubber latex allergy is still important, but increased risks of chlorhexidine and ethylene oxide allergy could not be confirmed.