Introduction: This study aimed to assess the impact of a stethoscope disinfection sensitization campaign among doctors and nurses in a Nigerian teaching hospital. Methodology: The design was a before-and-after study. Pre-program measurements were used to provide a baseline against which the post-program results were compared. Interventions that promoted compliance with stethoscope disinfection practice that were implemented included training and education on stethoscope disinfection and introduction of 70% isopropyl alcohol disinfectant at points-of-care places. Microbiological assessment of stethoscopes used by health workers was conducted after the intervention and the outcome was compared with the pilot study results. Results: After the intervention, of the 89 stethoscopes screened, 18 (20.2%) were contaminated with bacterial agents. A higher prevalence of stethoscope contamination was observed among stethoscopes from the intensive care unit (66.7%), the VIP unit (50%), and the antenatal unit (37.5%). The main isolates were Staphylococcus aureus (44.4%) and Escherichia coli (50%). The antibiotic sensitivity assessment indicated that the bacterial isolates were resistant to nearly all the antibiotics tested. All the 89 health workers whose stethoscopes were screened after the intervention admitted to cleaning their stethoscopes after seeing each patient, representing a compliance rate of 100%, unlike the 15% compliance at the pilot phase. The baseline stethoscope contamination rate was 78.5% versus 20.2% post-intervention. Conclusions: Training and education and introduction of alcohol-based disinfectants inexpensive but very effective methods to improve stethoscope disinfection compliance among health workers in low-income settings.