Disability is negatively associated with the health of older adults, and it can be mediated by healthy lifestyles and behaviors throughout one's life. In this context, understanding the interrelationships between sedentary behavior, physical activity and functionality may assist in the implementation of effective public health actions. Thus, the aim of the present study was to investigate the relationships between both physical activity and sedentary behavior and functionality in older adults and the possible mediators. The variables analyzed were selected according to the content analysis of International Classification of Functioning, Disability and Health model, and included activity, participation, health conditions, body functions and structures, environmental factors and personal factors. 419 individuals participated in the study. Physical activity was directly associated with disability in instrumental activities of daily living (IADL), and the association was mediated by self-esteem, aerobic endurance, and agility/balance. Sedentary behavior was indirectly associated with IADL disability, and the association was mediated by aerobic resistance, nutritional status, and agility/balance. Regarding the basic activities of daily living (BADL), physical activity showed an indirect association mediated by aerobic resistance and IADL. The association of sedentary behavior with BADL was mediated by aerobic resistance and lower limb flexibility. These results reinforce the idea that functionality is multidimensional, and the mediating factors must be considered when strategies for promoting physical activity and reducing sedentary behavior are designed.