Two challenges in topical drug delivery to the skin include solubilizing hydrophobic drugs in water-based formulations and increasing drug penetration into the skin. Polymeric core-multishell nanocarrier (CMS), particularly the novel biodegradable CMS (bCMS = hPG-PCL1.1K-mPEG2k-CMS) have shown both advantages on excised skin ex vivo. Here, we investigated topical delivery of tacrolimus (TAC; > 500 g/mol) by bCMS in a hydrogel on an oxazolone-induced model of dermatitis in vivo. As expected, bCMS successfully delivered TAC into the skin. However, in vivo they did not increase, but decrease TAC penetration through the stratum corneum compared to ointment. Differences in the resulting mean concentrations were mostly non-significant in the skin (epidermis: 35.7 ± 20.9 ng/cm2 for bCMS vs. 92.6 ± 62.7 ng/cm2 for ointment; dermis: 76.8 ± 26.8 ng/cm2vs 118.2 ± 50.4 ng/cm2), but highly significant in blood (plasma: 1.1 ± 0.4 ng/ml vs 11.3 ± 9.3 ng/ml; erythrocytes: 0.5 ± 0.2 ng/ml vs 3.4 ± 2.4 ng/ml) and liver (0.01 ± 0.01 ng/mg vs 0.03 ± 0.01 ng/mg). bCMS were detected in the stratum corneum but not in viable skin or beyond. The therapeutic efficacy of TAC delivered by bCMS was equivalent to that of standard TAC ointment. Our results suggest that bCMS may be a promising carrier for the topical delivery of TAC. The quantitative difference to previous results should be interpreted in light of structural differences between murine and human skin, but highlights the need as well as potential methods to develop more a complex ex vivo analysis on human skin to ensure quantitative predictive value.