Multidrug-resistant tuberculosis (MDR-TB) is one of the major public health concerns worldwide particularly in developing countries, including Bangladesh. Thus far, there are no well-validated clinical guidelines for the prevention of MDR-TB. This study aims to evaluate the improvement in nurses' practice using the newly developed Nursing Practice Guidelines for the Prevention of MDR-TB (NPG: MDR-TB) among hospitalized adult patients in Bangladesh. The guidelines were developed, disseminated and evaluated among 64 nurses by assessing nursing practice for the prevention of MDR-TB during pre- and postimplementation of the guidelines. Significant differences between pretest and post-test mean scores of nursing practice for the prevention of MDR-TB in case finding and case holding were found in three levels of wards, including Level 0 (non-TB), Level 1 (TB) and Level 2 (MDR-TB) (P < 0.001). This indicated that the guidelines might be applicable to reduce the development of MDR-TB in hospitals. However, this was a preliminary study with a limited time frame. Further evaluation is, therefore, needed.