Abstract Introduction Prescription of potentially inappropriate medications, non-geriatric doses and drug-disease interactions contribute to high prevalence of adverse drug events, geriatric syndromes and symptoms, and increased frailty in older adults. Despite serious health and socio-economic consequences, a very few studies have been published on phenomenon of prescribing of drug-disease interactions (DDI) in older patients in Central and Eastern Europe. Methods This study aimed to investigate the prevalence of DDI and differences across nursing home (NH) facilities applying relevant parts of Beers 2012 criteria, Czech national consensus 2012 (CNC), and STOPP/START criteria vers.1. This is a retrospective cross-sectional study that analysed semi-implicitly InterRAI-LTC assessment protocols of 490 NH residents from 10 Czech NH facilities (N = 490, 65+) participating in the EU SHELTER project. Retrospective analyses were conducted in 2019 year. Results Prevalence of potentially inappropriate DDI ranged from 44.5% to 62.3% identified by STOPP criteria and CNC, respectively. The most common DDIs were long-term use of benzodiazepines in depressive residents (7.8%) and use of opioids in residents with chronic constipation without osmotic laxative treatment (7.4%). The prevalence of undertreatment identified by START criteria was 52.9%, mainly due to absence of statins in NH residents with diabetes mellitus and cardiovascular risk factors (9.8%) and the absence of anticoagulation therapy in patients with atrial fibrillation (7.1%). Conclusions Potentially inappropriate DDI were highly prevalent in Czech long-term NH residents with significant differences across NH facilities. Using method of semi-implicit medication reviews, we cannot judge the real quality of drug treatment, but there is a necessity to reduce the high prevalence of DDI in NHs in order to prevent potential adverse drug events. Grant support: InoMed project (reg. No: CZ.02.1.01/0.0/0.0/18_069/0010046, 2019–2022), H2020-MCSF-ITN-764632, PROGRESS Q42 FoP, Charles University, FP7-HEALTH-F4–2008-201,917, SVV 260417.