Background: Mobile health (mHealth) apps are increasingly used to support healthy lifestyle behaviors through features such as health tracking and personalized reminders. Personalized messaging, tailored to users' profiles, has been shown to improve engagement and retention in health-related contexts. Prior research has linked personality traits, based on the Big Five model, to preferences for specific app mechanisms, leading to the development of a preference matrix for personalizing mHealth apps. This matrix comprises 15 mechanisms derived from behavior change techniques and gamification elements, intended to guide developers in optimizing engagement according to user profiles. Objective: This study aimed to validate this preference matrix by examining whether the associations between mechanisms and Big Five personality traits reported in the literature align with user preferences observed in an experimental setting. Methods: A cross-sectional study was conducted using an online survey that collected demographic data, mHealth app usage, and personality traits. Participants were presented with mockups illustrating 15 mechanisms and were asked to select their preferred options. Logistic regression and ordinal logistic regression analyses were performed to examine associations between personality traits, mechanism selection, and motivation scores. All analyses were adjusted using the Bonferroni correction to account for multiple comparisons. Results: A total of 214 participants completed the survey (mean age 29.42, SD 10.41 y; n=118, 55.1% women; n=89, 41.6% men; n=5, 2% identifying as other; and n=2, 1% nonrespondents). Higher conscientiousness significantly increased the likelihood of selecting the collection mechanism (eg, collecting badges or points; odds ratio [OR] 1.87, 95% CI 1.27-2.75). For competition (eg, competing with other users), conscientiousness (OR 3.22, 95% CI 1.73-6.00) and agreeableness (OR 1.93, 95% CI 1.08-3.45) were significant predictors. Preferences for rewards (eg, virtual incentives such as points or virtual currency) were associated with conscientiousness (OR 2.36, 95% CI 1.53-3.63) and neuroticism (OR 1.97, 95% CI 1.36-2.86). Additionally, 4 mechanisms-self-monitoring, progression, challenge, and quest-were selected by more than half of the participants, independent of personality traits. Conclusions: The findings partially validate the proposed preference matrix. Conscientiousness consistently emerged as a key predictor of preference across multiple mechanisms, highlighting its central role in engagement with gamified mHealth features. While some mechanisms appear to have universal appeal, others show personality-specific preferences, underscoring the value of combining baseline mechanisms with targeted personalization strategies in mHealth app design.