Background Diffuse implementation of hepatitis C virus (HCV) treatment is dependent on universal screening for HCV, but screening strategies are heterogenous across prisons in the province of Quebec (Canada). We sought to identify barriers and enablers to universal opt-out HCV screening and to describe the multisectoral decision-making processes related to HCV screening in Quebec provincial prisons. Methods A multilevel, multi-theory informed qualitative descriptive approach was used to conduct semi-structured interviews. Interview guides and analyses with correctional stakeholders were informed by the Consolidated Framework for Implementation Research (CFIR) and those with healthcare professionals (HCPs) were based on the Theoretical Domains Framework (TDF). Directed content analysis was used to identify domains within CFIR and TDF reflecting barriers and enablers to opt-out HCV screening. Results Sixteen interviews (correctional stakeholders: n = 8; HCPs: n = 8) were conducted in April-May 2021. Twelve CFIR constructs were identified as barriers, seven as enablers, and two as neutral factors for the implementation of opt-out HCV screening. Correctional stakeholders underscored the need for political will (construct: external policy and incentives), highlighted limited resources (construct: available resources), and expressed concerns for the lack of consideration of implementation issues (constructs: trialability, planning). Six TDF domains were identified among HCPs as relevant to the implementation of opt-out HCV screening: beliefs about consequences (mixed = enablers and barriers), environmental context and resources (barrier), social influences (barrier), optimism (mixed), emotions (mixed), and behavioural regulation (barrier). The decision-making processes vis-à-vis HCV care in Quebec correctional settings were found to be hierarchical and complex. Conclusions The use of CFIR and TDF was helpful in identifying barriers and enablers to HCV screening at multiple levels for people incarcerated in Quebec provincial prisons. Going forward, several political, structural, and organizational factors should be addressed through the engagement of stakeholders and people with lived experience of incarceration.