Introduction Properly educated nurses delivering evidence-based tobacco dependence treatment could contribute to improving health and reducing regional disparities in tobacco-related disease and death in Central and Eastern Europe. The aims of this study are to describe development of the Eastern European Nurses’ Centre of Excellence for Tobacco Control (COE) and evaluate its online educational program on tobacco dependence treatment using the 5As framework. Methods The online education evaluation followed a prospective, single group, pre- and post-assessment of changes in nurses’ self-reported tobacco cessation interventions. Leaders from five Eastern European countries (Hungary, Czech Republic, Romania, Slovakia, Slovenia) developed protocols for in-country tobacco control education. Nurses responded to a baseline survey, accessed an online nursing educational program, and completed a follow-up survey at 3 months, in the period December 2015 to June 2016. A total of 695 nurses from five countries answered questions on cessation interventions at baseline and of these 507 (73%) completed a follow-up survey at 3 months. Results At the follow-up at 3 months, the 507 nurses self-reported a significant increase in providing all 5A components, i.e. nurses were significantly (p<0.0001) more likely to always /usually advise a patient to quit, assess interest in quitting (p=0.002), assist with a quit plan, review barriers to quitting and recommend a smoke-free home post-discharge (all p<0.0001). They were also significantly more likely (p=0.01) to agree or strongly agree that nurses have an obligation to advise patients on the risks of smoking. There was a significant increase (p<0.0001) in nurses’ estimate of the number of patients they provided a cessation intervention the previous week. Nurses who smoked were 60% more likely to assist and arrange compared to nurses who never smoked. Conclusions Coordinating multi-country activities through a COE was successful in engaging a network of nurses to use an online educational program and participate in other tobacco control activities.