Background Hair analysis is an important psychoneuroendocrine method for assessing long-term hormonal activity, yet determinants of hair steroid concentrations in early infancy remain understudied. This study evaluated the influence of developmental (sociodemographic and birth-related) as well as methodological and contextual (hair-related, organizational, and COVID-19 exposure) factors on hair steroids during the first two years of life. Methods Children's hair samples from the prospective DREAM_HAIR study were collected at three weeks ( n = 210), eight weeks ( n = 240), 14 months ( n = 245), and two years ( n = 219). Cortisol (HairF), cortisone (HairE), dehydroepiandrosterone (HairDHEA), progesterone (HairP), testosterone (HairT), and corticosterone (HairB) were quantified in the proximal 2 cm hair segment using liquid chromatography–tandem mass spectrometry. Results HairF, HairE, and HairP peaked postnatally, declined sharply within eight weeks, and decreased gradually thereafter. HairF was strongly correlated with HairE (T3–T4), while HairP and HairDHEA showed moderate to strong correlations with both HairF and HairE (T2–T4). Regression analyses revealed associations with sex (HairDHEA), hair color (HairP), season (HairE, HairDHEA), storage time (HairDHEA), gestational week (HairF), and birth mode (HairF). After correction for multiple testing, only birth mode (HairF, T1) and season (HairDHEA, T4) remained significant. Body weight and COVID-19 exposure were not related to hair steroids. High non-detectable rates for HairT and HairB limited analyses to descriptive statistics. Conclusion Developmental, methodological and contextual influences on hair steroids were small, with consistent effects of birth mode, season, and age-related changes. Hair analysis appears to be a promising method for assessing long-term endocrine activity in early childhood.