Background: Body-worn sensors allow assessment of gait characteristics that are predictive of fall risk, both when measured during treadmill walking and in daily life. The present study aimed to assess differences as well as associations between fall-related gait characteristics measured on a treadmill and in daily life. Methods: In a cross-sectional study, trunk accelerations of 18 older adults (72.3 ± 4.5 years) were recorded during walking on a treadmill (Dynaport Hybrid sensor) and during daily life (Dynaport MoveMonitor). A comprehensive set of 32 fall-risk-related gait characteristics was estimated and compared between both settings. Results: For 25 gait characteristics, a systematic difference between treadmill and daily-life measurements was found. Gait was more variable, less symmetric, and less stable during daily life. Fourteen characteristics showed a significant correlation between treadmill and daily-life measurements, including stride time and regularity (0.48 < r < 0.73; p < 0.022). No correlation between treadmill and daily-life measurements was found for stride-time variability, acceleration range and sample entropy in vertical and mediolateral direction, gait symmetry in vertical direction, and stability estimated as the local divergence exponent by Rosenstein's method in mediolateral direction (r < 0.16; p > 0.25). Conclusions: Gait characteristics revealed less stable, less symmetric, and more variable gait during daily life than on a treadmill, yet about half of the characteristics were significantly correlated between conditions. These results suggest that daily-life gait analysis is sensitive to static personal factors (i.e., physical and cognitive capacity) as well as dynamic situational factors (i.e., behavior and environment), which may both represent determinants of fall risk.