Background <p>Menstrual hygiene is essential for adolescent girls, as it greatly affects their capacity to engage in academic and extracurricular activities. Adolescent girls often encounter various challenges in maintaining proper menstrual hygiene, making it essential to explore these challenges to design effective future interventions. Therefore, this study was conducted to assess the knowledge, practices, perceived challenges, and factors associated with menstrual hygiene among adolescent school girls in Galle Educational Zone in southern Sri Lanka.</p> Methods <p>This study employed a mixed-methods design, utilizing a sequential explanatory strategy, comprising a quantitative descriptive component followed by an exploratory descriptive qualitative component. The study was conducted with the participation of 389 girls in the Galle educational division using the multi-stage cluster sampling method in 2024. A validated, modified, self-administered questionnaire was used to collect data. Descriptive statistics, Chi-square test, independent sample t-test, one-way ANOVA, and multiple linear regression were used based on the type of variable. For the qualitative component, in-depth interviews were conducted with 15 purposively selected school girls. The interviews were transcribed verbatim, and the data were coded systematically to identify recurring patterns and themes. The ethical approval was obtained from the ethics review committee Faculty of Allied Health Sciences, University of Ruhuna.</p> Results <p>Among the participants, only 32.7% (<i>n</i> = 125) had satisfactory knowledge, and only 17.3% (<i>n</i> = 66) had a satisfactory level of practice on menstrual hygiene. Knowledge on menstruation was associated with menstrual hygiene practices (<i>p</i> = 0.015), information received from the media (<i>p</i> = 0.010), experience on menstruation (<i>p</i> = 0.025), mother’s level of education (<i>p</i> &lt; 0.001) and father’s level of education (<i>p</i> &lt; 0.001). Hygiene practices on menstruation were associated with the type of family (<i>p</i> = 0.023). In the qualitative component, under the perceived challenges, four sub-themes emerged: comfort and dignity, socio-cultural taboos, physical constraints, and insufficient resources.</p> Conclusion <p>The participants' overall knowledge on menstruation and menstrual hygiene was inadequate. Key factors influencing menstrual hygiene included parents' level of education, family structure, and prior experience with menstruation.</p>