Background <p>Many studies have highlighted the association between frailty, physical activity behaviour (PAB), and well-being, but no study has investigated a potential moderated mediating role of functional difficulty in this relationship. This association may not be the same between different income levels. This study, therefore, assessed the above moderated mediation between low- and higher-income samples.</p> Methods <p>This research employed a cross-sectional design in accordance with established research-reporting guidelines. The study population comprised two distinct Ghanaian samples, with <i>N</i> = 942 individuals in the low-income group and <i>N</i> = 600 individuals in the higher income group. Data analysis was carried out using Hayes's Process model through structural equation modelling, with additional sensitivity analyses performed through hierarchical linear regression.</p> Results <p>Frailty had a direct negative effect and an indirect negative effect (through functional difficulty) on well-being in both samples. A partial mediation of functional difficulty was found in the relationship between frailty and well-being in both samples. We also found evidence of a moderated mediation by functional difficulty in both samples; however, this effect was stronger in the higher-income sample.</p> Conclusion <p>Older employees with frailty are less likely to report lower functional difficulty and well-being at higher PAB. Our results suggest a need for workplace programmes aimed at encouraging PAB. It also reinforces the importance of individuals performing and maintaining PAB.</p>