Healthcare workers are at increased risk of contracting hepatitis B virus (HBV), particularly in settings of high HBV seroprevalence, such as sub-Saharan Africa. We evaluated HBV knowledge among health-care workers in rural Tanzania by distributing an HBV paper survey in two northern Tanzanian hospitals. There were 114 participants (mean age 33 years, 67% female). Of the participants, 91% were unaware of their HBV status and 89% indicated they had never received an HBV vaccine, with lack of vaccine awareness being the most common reason (34%), whereas 70% were aware of HBV complications and 60% understood routes of transmission. There was a significant difference in knowledge of HBV serostatus and vaccination between participants with a medical background and others, P = 0.01 and 0.001, respectively. However, only 33% of consultants (senior medical staff) knew their HBV serostatus. There was no significant difference between knowledge of HBV transmission routes and occupation. Our study reveals low knowledge of HBV serostatus and vaccination status among hospital workers in Tanzania.