We evaluated the impact of infection control interventions to reduce nosocomial extended-spectrum beta-lactamase (ESBL) transmission in a non-outbreak setting. This study was conducted at a tertiary 1200-bed hospital in Canada. The incidence of ESBLs was based on recovery of clinical isolates and assessed prospectively from 1999 to 2005. The incidence increased significantly from 0.28 to 0.67 per 1000 admissions during this period (P<0.001), reflecting an increase in the regional ESBL incidence from 1.32 to 9.28 per 100 000 population (P<0.001). Despite this increase, nosocomial ESBL rates increased only marginally, suggesting that infection control measures had an impact on nosocomial transmission. Infection control measures consisted of isolating all ESBL patients, as well as implementing the use of contact precautions for those with a high risk for transmission. The cost of these measures was CN$138 046.00 per year and CN$3191.83 per case admitted. A combination of control measures including active surveillance cultures, contact precautions for all colonized or infected patients and antimicrobial stewardship is required to significantly reduce the incidence of ESBLs.