Background: There is a gap in the research about the influence of wearing or not wearing a prosthesis for muscle strength assessment in transfemoral amputees (TFA) and how it is associated with functional mobility. Objective(s): The aim of this study was to compare the muscle isometric strength of the residual limb with and without the prosthesis in people with TFA and to analyze associations between muscle strength and functional mobility. Study Design: Cross-sectional study. Methods: 20 subjects with TFA were included. A handheld dynamometer was used for the assessment of residual limb muscle strength. Functional mobility was assessed with the Timed Up and Go test. The Wilcoxon rank sum test with the rank biserial correlation effect size were used. Results: There were statistically significant differences when testing isometric strength of the residual limb with and without the prosthesis (flexion [p = 0.007], extension [p < 0.001], and abduction [p = 0.003]). There was association between functional mobility and flexion and abduction strength with the prosthesis (p = 0.005, p = 0.01). Conclusions: Measurements of muscle strength of the residual limb were different when assessed with and without the prosthesis. Isometric strength of the residual limb in abduction and flexion using the prosthesis were correlated with functional mobility.