Background and objective. Wearing of gloves reduces transmission of organisms by healthcare workers’ hands but is not a substitute for hand hygiene. Results of previous studies have varied as to whether hand hygiene is worse when gloves are worn. Most studies have been small and used non-standardized assessments of glove use and hand hygiene. We sought to observe whether gloves were worn when appropriate and whether hand hygiene compliance differed when gloves were worn. Design. Observational study. Participants and setting. Healthcare workers in 56 medical or care of the elderly wards and intensive care units in 15 hospitals across England and Wales. Methods. We observed hand hygiene and glove usage (7,578 moments for hand hygiene) during 249 one-hour sessions. Observers also recorded whether gloves were or were not worn for individual contacts. Results. Gloves were used in 1,983 (26.2%) of the 7,578 moments for hand hygiene and in 551 (16.7%) of 3,292 low-risk contacts; gloves were not used in 141 (21.1%) of 669 high-risk contacts. The rate of hand hygiene compliance with glove use was 41.4% (415 of 1,002 moments), and the rate without glove use was 50.0% (1,344 of 2,686 moments). After adjusting for ward, healthcare worker type, contact risk level, and whether the hand hygiene opportunity occurred before or after a patient contact, glove use was strongly associated with lower levels of hand hygiene (adjusted odds ratio, 0.65 [95% confidence interval, 0.54–0.79]; ). P < .0001 Conclusion. The rate of glove usage is lower than previously reported. Gloves are often worn when not indicated and vice versa. The rate of compliance with hand hygiene was significantly lower when gloves were worn. Hand hygiene campaigns should consider placing greater emphasis on the World Health Organization indications for gloving and associated hand hygiene. Trial registration. National Research Register N0256159318.