Background Research suggests that physical activity (PA) has both acute and chronic beneficial effects on cognitive function in laboratory settings and under supervised conditions. Mobile health technologies make it possible to reliably measure PA and cognition in free-living environments, thus increasing generalizability and reach. Research is needed to determine whether the benefits of PA on cognitive function extend from the laboratory to real-world contexts. Objective This observational study aims to examine the association between daily fluctuations in PA and cognitive performance using mobile health technologies in free-living environments. Methods A total of 90 adults (mean age 59, SD 6.3 years; 65/90, 72% men) with various comorbidities (eg, cardiovascular risk and HIV) and different levels of baseline cognition (ranging from cognitively normal to impaired) completed ecological momentary cognitive tests (EMCTs) on a smartphone twice daily while wearing an accelerometer to capture PA levels for 14 days. Linear mixed-effects models examined the daily associations of PA with executive function and verbal learning EMCTs. Moderation analyses investigated whether the relationship between daily PA and daily performance on EMCTs changed as a function of baseline cognition, cardiovascular risk, and functional status (independent vs dependent). Results Days with greater PA were associated with better (faster) performance on an executive function EMCT after covariate adjustment (estimate −0.013; β=−.16; P=.04). Moderation analyses (estimate 0.048; β=.58; P=.001) indicated that days with greater PA were associated with better (faster) executive function performance in individuals who were functionally dependent (effect size −0.53; P<.001) and not in functionally independent adults (effect size −0.01; P=.91). Conclusions EMCTs may be a sensitive tool for capturing daily-level PA-related fluctuations in cognitive performance in real-world contexts and could be a promising candidate for tracking cognitive performance in digital health interventions aimed at increasing PA. Further research is needed to determine individual characteristics that may moderate the association between daily PA and EMCT performance in free-living environments.