Abstract Background Institutional mistrust, the lack of confidence and overall suspicion towards authorities or systems, usually governmental, but also extending to the health sector, poses a major obstacle towards public health policies and actions. Lower educational attainment has been linked to higher mistrust, but the role of health literacy in particular remains unclear. Methods Cross-sectional study in the general adult population of Greece and Cyprus following proportional quota sampling (January 2022 to May 2023). The current analysis is based on a sub-sample of 292 participants, with complete information on health literacy (main exposure, assessed via the validated HLS-EU-Q16) and health-related institutional mistrust (main outcome, assessed via a new culturally adopted questionnaire comprising 11 items). Multiple linear regression was used to determine associations between the aforementioned factors, adjusting for educational attainment and other potential confounders. Results Exploratory factor analysis revealed 3 health-related institutional mistrust dimensions, towards: (a) governmental authorities and the media (i.e., the system), (b) doctors and other health professionals, and (c) the scientific community. Health literacy was negatively associated with all mistrust dimensions, with associations being particularly strong with mistrust towards the system (B, 95% CI per unit increase in the HL scale: -0.87, -1.58; -0.15) and the scientific community (-0.71, -1.11; -0.32). These association remained robust to adjustment for educational attainment and other sociodemographic factors. Conclusions Mistrust towards a perceived system, comprising the government and media, as well as towards the scientific community on health-related matters, is higher among individuals with low health literacy, irrespective of educational attainment. Enhancing health literacy could act as a novel target for public health approaches aiming to tackle health-related institutional mistrust. Key messages • Health-related institutional mistrust is a complex phenomenon, which appears to have different dimensions, relevant to the perceived system, health professionals, and the scientific community. • Low health literacy is an independent predictor of health-related institutional mistrust, rendering it a novel target for attempts towards reversing this phenomenon and its adverse health consequences.