Background Older adults admitted with an acute medical illness spent little time active during hospitalisation and this has been associated with negative health outcomes. Understanding which barriers and enablers influence the physical activity behaviour of hospitalised older adults is a first step towards identifying potentially modifiable factors and developing, evaluating and implementing targeted interventions aimed at increasing their physical activity behaviour. Using a theoretical framework has been found to be more successful in changing behaviour than using a non-theory driven approach. This study aimed to explore barriers and enablers to physical activity behaviour in older adults admitted to a hospital with an acute medical illness, as perceived by patients and healthcare professionals, and to categorise them using the Theoretical Domains Framework (TDF). Methods A qualitative study was conducted at a combined university and regional hospital in the Netherlands between January 2019 and February 2020. Older adults (≥70 years) admitted with an acute medical illness, and healthcare professionals (nurses, physicians, physiotherapists) were recruited using purposive sampling. Semi-structured interviews were audiotaped, transcribed and analysed using directed qualitative content analysis. Barriers and enablers to physical activity behaviour during hospitalisation were identified and coded using the TDF. Results Meaning saturation was determined after interviews with 12 patients and 16 healthcare professionals. A large number of barriers and enablers were identified and each categorised to 11 of the 14 domains of the TDF. The ‘Environmental Context and Resources’ domain in particular yielded many examples, and revealed that the hospital environment exerts an inactivating influence on patients. Conclusions The large number of identified barriers and enablers highlights the complexity of influencing older adults’ physical activity behaviour during hospitalisation. This overview of barriers and enablers to physical activity behaviour in older adults admitted to a hospital with an acute medical illness represents an initial step towards developing, evaluating and implementing theory-informed behaviour change interventions to improve hospitalised older adults’ physical activity levels. It can assist clinicians and researchers in selecting modifiable factors that can be targeted in future interventions.