Abstract Background Tailored and culturally appropriate latent tuberculosis (TB) infection screening and treatment programs, including interventions against TB stigma, are needed to reduce TB incidence in low TB incidence countries. However, we lack insights in stigma related to latent TB infection (LTBI) among target groups, such as asylum seekers and refugees. We therefore studied knowledge, attitudes, beliefs, and stigma associated with LTBI among Eritrean asylum seekers and refugees in the Netherlands. Methods We used convenience sampling to interview adult Eritrean asylum seekers and refugees: 26 semi-structured group interviews following TB and LTBI related health education and LTBI screening, and 31 semi-structured individual interviews with Eritreans during or after completion of LTBI treatment (November 2016–May 2018). We used a thematic analysis to identify, analyse and report patterns in the data. Results Despite TB/LTBI education, misconceptions embedded in cultural beliefs about TB transmission and prevention persisted. Fear of getting infected with TB was the cause of reported enacted (isolation and gossip) and anticipated (concealment of treatment and self-isolation) stigma by participants on LTBI treatment. Conclusion The inability to differentiate LTBI from TB disease and consequent fear of getting infected by persons with LTBI led to enacted and anticipated stigma comparable to stigma related to TB disease among Eritreans. Additional to continuous culturally sensitive education activities, TB prevention programs should implement evidence-based interventions reducing stigma at all phases in the LTBI screening and treatment cascade.