INTRODUCTION: Over the past two decades, Tanzania's burden of non-communicable diseases has grown disproportionately, but limited resources are still prioritized. A trained human resource for health is urgently needed to combat these diseases. However, continuous medical education for NCDs is scarce. This paper reports on the mid-level healthcare workers knowledge on NCDs. We assessed the knowledge to measure the effectiveness of the training conducted during the initiation of a Package for Essential Management of Severe NCDs (PEN Plus) in rural district hospitals in Tanzania. METHODS: The training was given to 48 healthcare employees from Dodoma Region's Kondoa Town Council District Hospital. For a total of five (5) days, a fundamental course on NCDs featured in-depth interactive lectures and practical workshops. Physicians from Tanzania's higher education institutions, tertiary university hospitals, research institutes, and medical organizations served as trainers. Before and after the training, a knowledge assessment comprising 28 questions was administered. Descriptive data analysis to describe the characteristics of the specific knowledge on physiology, diagnosis and therapy of diabetes mellitus, rheumatic fever, heart disease, and sickle cell disease was done using Stata version 17 (STATA Corp Inc., TX, USA). RESULTS: Complete assessment data for 42 out of the 48 participants was available. Six participants did not complete the training and the assessment. The mean age of participants was 36.9 years, and slightly above half (52%) were above 35 years. Two-thirds (61.9%) were female, and about half (45%) were nurses. The majority had the experience of working for more than 5 years, and the average was 9.4 years (+/- 8.4 years). Overall, the trainees' average scores improved after the training (12.79 vs. 16.05, p < 0.0001) out of 28 possible scores. Specifically, trainees' average scores were better in treatment than in diagnosis, except for sickle cell disease (1.26 vs. 1.83). Most were not able to diagnose rheumatic heart disease (47.6% able) compared to diabetes mellitus (54.8% able) or sickle cell disease (64.3% able) at baseline. The proportion of trainees with adequate knowledge of the treatment of sickle cell disease and diabetes mellitus was 35% and 38.1%, respectively, and there was a non-statistical difference after training. Those working for less than 5 years had a higher proportion of adequate knowledge (30.8%) compared to their more experienced colleagues (6.9%). After the training, participants' knowledge of NCDs increased by three times (i.e., aPR 3, 95% CI = 1.1, 1.5, and 6.0). CONCLUSION AND RECOMMENDATIONS: PEN Plus training improved the knowledge of healthcare workers at Kondoa Town Council District Hospital. Training is especially needed among nurses and those with a longer duration of work. Continuing education for human resources for health on the management of NCDs is highly recommended in this setting.