We determined prospectively the frequency, persistence and molecular epidemiology of Clostridium difficile environmental contamination after detergent-based cleaning in side rooms used to isolate patients with C. difficile diarrhoea. Approximately one-quarter of all environmental sites in side rooms sampled over four-week periods were contaminated with C. difficile. The overall side room prevalence of environmental C. difficile declined from 35% initially, to 24% in week 2, 18% in week 3, and 16% in week 4. The bed frame was the most common site from which C. difficile was recovered, although the floor was the most contaminated site in terms of total numbers of colonies. C. difficile was recovered significantly more frequently from swabs plated directly on to C. difficile selective media containing lysozyme than from enrichment broth (P < 0.001), emphasizing the benefit of lysozyme supplementation. The great majority of C. difficile isolates (87% of all isolates, 84% of patient isolates) was indistinguishable from the UK epidemic strain (PCR ribotype 1). It thus could not be determined whether environmental contamination was a cause or a consequence of diarrhoea. Our findings highlight the need for improved approaches to hospital environmental hygiene, and call into question current UK guidelines that recommend detergent-based cleaning to remove environmental C. difficile. In particular, improved cleaning of frequently touched sites in the immediate bed space area is required.