The rate of healthcare-associated infections can be regarded as an important outcome parameter of the hygienic quality of care in nursing homes. Our study aimed to evaluate the applicability of repeated prevalence investigations as a tool for surveillance of healthcare-associated infections in nursing homes. From December 2006 to September 2007 a total of five prevalence investigations were conducted in four nursing homes each (n=2,369 residents). Initially, defined structural and procedural parameters of the hygienic quality of the four nursing homes were evaluated based on a detailed inspection and a checklist including 40 parameters. The results showed a uniformly high level of the hygienic quality with only minor variation (mean 84%, range 75%-93% of parameters fulfilled). In total, the prevalence of healthcare-associated infections was 6.8%, with a marked increase with higher categories of dependency (3.5%, 4.0%, 8.5%, and 12.3%, respectively, in the categories 0, I, II, and III of the German grading of skilled nursing care). Respiratory tract (4.1%), skin/soft tissue (1.5%), and urinary tract infections were the most prevalent healthcare-associated infections. Respiratory tract infections showed a marked seasonal pattern. During the second prevalence investigation (February 2007), an outbreak of upper respiratory tract infections occurred in one of the nursing homes (attack rate, 17%). The crude prevalence rates showed considerable differences between the four nursing homes; however, after adjusting for the different categories of dependency, the standardized infection rates (SIR) were largely comparable (excluding the outbreak). After inclusion of the outbreak, the SIR of the specific nursing home was significantly higher compared to all other nursing homes. In conclusion, our study shows that repeated prevalence investigations can be an easy to use tool for surveillance of healthcare-associated infections as a surrogate parameter of the hygienic quality in nursing homes. This implies a knowledge of the seasonality of specific infections and a risk adjustment according to the categories of dependency. The primary intention of surveillance should be the identification of hygienic problems. However, the resources should preferentially be focused on hygienic structures and processes.