Improvement in hand hygiene compliance is important for reducing cross-infection by micro-organisms. The objective of this prospective observational study was to measure how the improper use of gloves limits compliance to hand hygiene and exposes patient's to infection. The study was conducted in five wards (three intensive care units and two medical wards) in a French university hospital. Staff–patient and staff–environment contacts were observed in 120 healthcare workers caring for patients colonized or infected with pathogenic bacteria. Hand hygiene was not undertaken due to improper gloving in 64.4% (95%CI, 64.1% to 65.1%) of instances. Possible microbial transmission might have occurred in 18.3% (95%CI, 17.8% to 18.8%) of all contacts because used gloves were not removed before performing care activities that necessitated strict aseptic precautions. Failure to change or remove contaminated gloves was a major component in the poor compliance with hand hygiene and carried a high-risk of microbial transmission. Improving hand hygiene compliance will require changing healthcare workers behaviour towards glove use.