Objectives: We evaluated the effect of an antibiotic stewardship programme (ASP) on the use of antibiotics and resistance levels of Escherichia coli using a method that allowed direct comparison between an intervention hospital and a control hospital. Methods: The study was conducted as a retrospective controlled interrupted time series (ITS) at two university teaching hospitals, intervention and control, with 736 and 552 beds, respectively. The study period was between January 2008 and September 2014. We used ITS analysis to determine significant changes in antibiotic use and resistance levels of E. coli. Results were directly compared with data from the control hospital utilizing a subtracted time series (STS). Results: Direct comparison with the control hospital showed that the ASP was associated with a significant change in the level of use of cephalosporins [2151 DDDs/1000 bed-days (95% CI 2177, 2126)] and fluoroquinolones [244.5 DDDs/1000 bed-days (95% CI 258.9, 230.1)]. Resistance of E. coli showed a significant change in slope for cefuroxime [20.13 percentage points/month (95% CI 20.21, 20.057)] and ciprofloxacin [20.15 percentage points/month (95% CI 20.26, 20.038)]. Conclusions: The ASP significantly reduced the use of cephalosporins and fluoroquinolones, with concomitant decreasing levels of E. coli resistance to cefuroxime and ciprofloxacin. The same development was not observed at the control hospital.