Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disorder in children, clinically characterized by intense pruritus and progressive lichenification. Cannabis sativa L. has been utilized as a medicinal plant since ancient times, serving as a remedy for various diseases in traditional medicine systems. C. sativa contains a variety of compounds, including flavonoids, terpenoids, and cannabinoids, which have antioxidant and anti-inflammatory effects. This study was conducted to investigate the antioxidant and anti-inflammatory effects of hybrid C. sativa inflorescence extracts (HCIE) in human keratinocytes, with a particular focus on elucidating their underlying molecular mechanisms. Compounds contained in HCIE were analyzed using UPLC, and intracellular ROS were evaluated using the DCF-DA staining method. Furthermore, HCIE-induced alterations in gene and protein expression levels were assessed using quantitative PCR and immunoblot analysis. A total of eight cannabinoids were identified in HCIE, among which cannabidolic acid exhibited the highest concentration. The gene expression of key chemokines associated with AD, including MDC, RANTES, and TARC, was suppressed in HCIE-treated HaCaT cells, accompanied by a reduction in their serum protein levels. In the HCIE-treated group, the phosphorylation of ERK, JNK, and p38 was downregulated, with phosphorylated JNK exhibiting the most pronounced reduction. In addition, nuclear translocation and phosphorylation of NF-κB were suppressed, and inhibition of NLRP3 and caspase-1 expression was also observed. On the other hand, the expression levels of skin barrier proteins, filaggrin and involucrin, were significantly upregulated in the HCIE-treated group, with filaggrin showing more than a twofold increase in the group treated with a high-concentration HCIE. Taken together, HCIE exerts anti-inflammatory and anti-atopic dermatitis effects by suppressing MAPK, NF-κB, and NLRP3 inflammasome activation, downregulating chemokine expression in keratinocytes, and upregulating skin barrier protein levels.