Vaccination is an important disease prevention and control measure; however, vaccine adoption by livestock farmers in Tanzania is still low. This cross-sectional study examined the challenges to vaccine use faced by livestock owners and animal health professionals (AHPs) in Tanzania. A questionnaire was administered to 216 households that kept small ruminants and poultry and 19 AHPs’ data were collected electronically via the survey platform Qualtrics, and descriptive statistics were performed. Households with poultry reported vaccinating mostly against Newcastle disease (91.7%), fowl pox (48.1%) and Gumboro disease (37.0%), whilst households with small ruminants reported contagious caprine pleuropneumonia (62.2%), sheep and goat pox (17.1%), foot-and-mouth disease (7.3%) and peste des petits ruminants (7.3%). The households’ decision to vaccinate was mostly influenced by knowledge of diseases (82.4%), disease history on the farm (69.4%) and vaccine price (63.4%). Most households (54.6%) experienced challenges when purchasing vaccines, including high vaccine cost (78.0%), long distance from vaccine source (61.0%) and vaccine unavailability (21.2%). The findings suggest that improving the knowledge of livestock owners regarding the priority diseases and the benefits of vaccination, establishing more vaccine suppliers, improving vaccine distribution and access and training AHPs and households on appropriate vaccine storage and handling are necessary to improve vaccine adoption and ensure vaccine quality and effectiveness.