An awareness of school-related antecedents of children’s physiological stress at the beginning of school helps educators to prevent and mitigate children’s stress, the one of the major obstacles to their well-being and academic progress. We aimed to study the effect of reading skills and social competence on first-grade students’ salivary cortisol levels in natural settings. Based on previous results of the effects of everyday situations on children’s stress according to gender, we expected that both academic and social skills would affect girls’ physiological stress more, compared to boys. Our sample consisted of 277 students (7–8 years old, 50.2% girls). We used the highest salivary cortisol level of three morning samples and a cortisol level from the middle of the school day as physiological stress indicators. Reading skills were assessed by group-administered tests and social competence by teacher ratings. We found that lower reading comprehension skills and lower disruptiveness were related to higher cortisol levels for girls but not for boys. Higher empathy and lower disruptiveness moderated the effect of better reading comprehension on higher psychological stress in the middle of the school day only for girls. By recognizing the antecedents of children’s stress and supporting their academic and social skills, children’s, especially girls’, physiological self-regulation and coping skills in the primary grades will benefit.