Atopic dermatitis (AD) is the most widespread chronic inflammatory skin disease characterised by impaired skin barrier, higher immunoglobulin E (IgE)-mediated sensitisation, and pronounced inflammatory and immune activity. In this study, mizolastine-loaded spanlastics (MZSPs) with edge activators (Brij 35, Tween 80, and Cremophor RH40) were formulated using ethanol injection method. Spanlastics were assessed for their average particle size, surface charge, encapsulation efficacy, morphology, in vitro drug release, and ex vivo skin permeation. MZSPs showed average particle size ranging from 186.2 ± 13.0 nm to 380.7 ± 25.9 nm and high MZ encapsulation efficiency percentage of (95.8 ± 0.68%-98.1 ± 0.86%). The prepared MZSPs were incorporated into 2.5% HPMC hydrogel base to facilitate its topical delivery. The in vitro release experiments showed a sustained release profile of MZ from MZSPs-hydrogel over 24 h with effective flux and permeation through rat skin compared to free drug. Additionally, the therapeutic efficacy of MZSPs-hydrogel in an AD model utilising seven-week-old male Balb/C mice outperformed the free drug in improving cytokine profiles, histopathological and immunohistochemical parameters, along with a quantitative analysis of mast cell immunostaining. These findings support the potential application of MZSPs for the transdermal delivery of MZ, offering enhanced management for AD.