Background Sustained high compliance with hand hygiene (HH) is needed to reduce nosocomial bloodstream infections (NBSIs). However, over time, a wash out effect often occurs. We studied the long-term effect of sequential HH-promoting interventions. Methods An observational study with an interrupted time series analysis of the occurrence of NBSI was performed in very low-birth weight (VLBW) infants. Interventions consisted of an education program, gain-framed screen saver messages, and an infection prevention week with an introduction on consistent glove use. Results A total of 1,964 VLBW infants admitted between January 1, 2002, and December 31, 2011, were studied. The proportion of infants with ≥1 NBSI decreased from 47.6%-21.2% (P < .01); the number of NBSIs per 1,000 patient days decreased from 16.8-8.9 (P < .01). Preintervention, the number of NBSIs per 1,000 patient days significantly increased by 0.74 per quartile (95% confidence interval [CI], 0.27-1.22). The first intervention was followed by a significantly declining trend in NBSIs of -1.27 per quartile (95% CI, -2.04 to -0.49). The next interventions were followed by a neutral trend change. The relative contributions of coagulase-negative staphylococci and Staphylococcus aureus as causative pathogens decreased significantly over time. Conclusions Sequential HH promotion seems to contribute to a sustained low NBSI rate.