Background: Conscious sedation is a useful adjunct in the treatment of patients in dentistry; however, a lack of knowledge among the dental profession regarding sedation is a restricting factor in the practice of dental sedation. Therefore, this study was conducted to assess the knowledge and practice of sedation in dentistry among dental professionals in Tanzania. Methods: This was a cross-sectional study conducted for five months targeting all practicing dental professionals in Tanzania. A modified questionnaire contained 14 questions regarding knowledge about sedative agents and a section on the practice of sedation. The data obtained from this study were coded and entered into a computer program and analyzed using SPSS software version 23.0. The data are presented as frequencies and percentages in tables and charts. Statistical significance was set at P < 0.05. Results: The age range of participants was between 24 and 63 years (mean 36.6 ± 7.7 years). There were 107 men (78.1%), and the male-to-female ratio was 3.6:1. The majority (76.6%) of participants only had an undergraduate dental degree. Thirty-one percent of participants only worked in publicly owned health facilities.Slightly more than half (59.9%) of participants had satisfactory knowledge regarding sedation in dentistry. There was no statistically significant association between the level of sedation-related knowledge and the demographic characteristics of the participants. Only 21.9% reported using sedation in their practice, and the most commonly used sedative drug was diazepam. The reasons for not using dental sedation in clinical practice included a perceived lack of knowledge on sedation, lack of equipment, and cost. Conclusion Most dental professionals in Tanzania have basic knowledge of sedation in dentistry, although knowledge regarding sedative agents is generally low. The practice of sedation in dentistry in Tanzania is very low compared to that in middle- and high-income countries. Inadequate knowledge, lack of equipment, and the cost of practicing sedation are the main reasons for not practicing sedation.