Background Vulvar squamous cell carcinoma (VSCC) is a rare disease with a poor prognosis. To date, there’s no proper in vitro modeling system for VSCC to study its pathogenesis or for drug evaluation. Methods We established healthy vulvar (HV)- and VSCC-like 3D full thickness models (FTMs) to observe the tumor-stroma interaction and their applicability for chemotherapeutic efficacy examination. VSCC-FTMs were developed by seeding VSCC tumor cell lines (A431 and HTB117) onto dermal matrices harboring two NF subtypes namely papillary fibroblasts (PFs) and reticular fibroblasts (RFs), or cancer-associated fibroblasts (CAFs) while HV-FTMs were constructed with primary keratinocytes and fibroblasts isolated from HV tissues. Results HV-FTMs highly resembled HV tissues in terms of epidermal morphogenesis, basement membrane formation and collagen deposition. When the dermal compartment shifted from PFs to RFs or CAFs in VSCC-FTMs, tumor cells demonstrated more proliferation, EMT induction and stemness. In contrast to PFs, RFs started to lose their phenotype and express robust CAF-markers α-SMA and COL11A1 under tumor cell signaling induction, indicating a favored ‘RF-to-CAF’ transition in VSCC tumor microenvironment (TME). Additionally, chemotherapeutic treatment with carboplatin and paclitaxel resulted in a significant reduction in tumor-load and invasion in VSCC-FTMs. Conclusion We successfully developed in vitro 3D vulvar models mimicking both healthy and tumorous conditions which serve as a promising tool for vulvar drug screening programs. Moreover, healthy fibroblasts demonstrate heterogeneity in terms of CAF-activation in VSCC TME which brings insights in the future development of novel CAF-based therapeutic strategies in VSCC. Graphic abstract