Research shows that poor knowledge and awareness of menstrual and pregnancy health among women are associated with adverse reproductive health and pregnancy outcomes. Menstrual cycle- and pregnancy-tracking mobile apps are promising tools for improving women's awareness of and attitudes toward their reproductive health; however, there is little information about subscribers' perceptions of app functionality and its impact on their knowledge and health. This study aimed to explore knowledge and health improvements related to menstrual cycle and pregnancy, as well as improvements in general health among Flo app users. We also investigated what components of the Flo app were associated with the abovementioned improvements and evaluated whether those improvements differed based on education level, country of residence (low- and middle-income vs high-income countries), free or premium subscription to the app, short- or long-term use of the app, and frequency of use. Flo subscribers who had been using the app for no less than 30 days, completed a web-based survey. A total of 2212 complete survey responses were collected. The survey included demographic questions and questions about motivations guiding the use of the Flo app and which components of the app improved their knowledge and health, as well as to what extent. Most study participants reported improvements in menstrual cycle (1292/1452, 88.98%) and pregnancy (698/824, 84.7%) knowledge from Flo app use. Participants with higher levels of education and those from high-income countries reported using the app predominantly for getting pregnant (χ21=4.2, P=.04; χ21=52.3, P<.001, respectively) and pregnancy tracking (χ21=19.3, P<.001; χ21=20.9, P=.001, respectively). Participants with less education reported using the app to avoid pregnancy (χ21=4.2; P=.04) and to learn more about their body (χ21=10.8; P=.001) and sexual health (χ21=6.3; P=.01), while participants from low- and middle-income countries intended to mainly learn more about their sexual health (χ21=18.2; P<.001). Importantly, the intended use of the app across education levels and country income levels matched areas in which they had gained knowledge and achieved their health goals upon use of the Flo app. Period, fertile days, and ovulation predictions as well as symptom tracking were consistently the top 3 components in the app that helped users with their cycle knowledge and general health. Reading articles or watching videos helped with users' education regarding their pregnancy. Finally, the strongest improvements in knowledge and health were observed in premium, frequent, and long-term users. This study suggests that menstrual health apps, such as Flo, could present revolutionary tools to promote consumer health education and empowerment on a global scale.