The effect of topical skin care products on neonatal skin barrier during first 8 weeks of life has not been scientifically evaluated. In a prospective, randomized clinical study, we compared the influence of three skin care regimens to bathing with water on skin barrier function in newborns at four anatomic sites. A total of 64 healthy, full-term neonates (32 boys and 32 girls) aged <48 hours were randomly assigned to four groups receiving twice-weekly: WG, bathing with wash gel (n = 16); C, bathing and cream (n = 16); WG + C, bathing with wash gel plus cream (n = 16); and B, bathing with water (n = 16). Transepidermal water loss, stratum corneum hydration, skin pH, sebum were measured on day 2, week 2, 4, 8 of life on front, abdomen, upper leg, and buttock. Skin condition was scored and microbiologic colonization was documented. After 8 weeks, group WG + C showed significantly lower transepidermal water loss on front, abdomen, and upper leg as well as higher stratum corneum hydration on front and abdomen compared with group B. Similarly, group C showed lower transepidermal water loss and higher stratum corneum hydration on these body regions. Group WG revealed significantly lower pH on all sites compared with group B at week 8. No differences in sebum level, microbiologic colonization and skin condition score were found. Skin care regimens did not harm physiologic neonatal skin barrier adaptation within the first 8 weeks of life. However, significant influence of skin care on barrier function was found in a regional specific fashion.