Objectives: The use of latex gloves has increased by several folds in the recent past due to concerns about blood-borne infections. Data from Asian countries with regard to latex allergy is scarce. The objective of this study was to determine the prevalence and risk factors of latex allergy among healthcare workers in a tertiary hospital in Sri Lanka. Material and Methods: A cross-sectional survey was carried out among different categories of employees in the hospital. A self-administered questionnaire was used to collect data related to latex allergy. Results: A total of 524 employees was recruited and 62% responded to the questionnaire. Among them 49.2% wore gloves for more than 1 hour a day. Symptoms suggestive of latex allergy were reported by 53 (16.3%) subjects. A considerable proportion (11.4%) of workers had been suffering from latex allergy for more than 5 years. Nurses accounted for the highest prevalence for any job category, while the unit with the highest rate was the surgical ward. Duration in the service (OR = 1.006, P = 0.048) and wearing gloves for more than one hour a day (OR = 3.292, P = 0.004) were significant risk factors for latex allergy, but not atopy or family history of atopy. Seven employees noticed that they developed food allergy after assuming duties as healthcare personnel. Conclusions: Prevalence of latex allergy is high among healthcare workers in this study population. Environmental factors rather than genetic predisposition play the major role in the development of this condition.