Despite advancements in therapeutic approaches, radiotherapy and cisplatin-based chemotherapy remain primary noninvasive treatments for patients with oral squamous cell carcinoma (OSCC). Moreover, the 5-year survival rate for patients with OSCC has remained almost unchanged for several decades, and many side effects of chemotherapy still exist. In this study, three-dimensional (3D) models of OSCC were established using fibroblasts, and the efficacy of various biological inhibitors was evaluated. A culture of epithelial cells with two types of fibroblasts (hTERT-hNOFs and cancer-associated fibroblasts) within a type I collagen matrix resulted in the formation of a continuous layer of tightly packed cells compared to models without fibroblasts. Furthermore, the effects of biological chemicals, including Y27632, latrunculin A, and verteporfin, on these models were investigated. The stratified formation of the epithelial layer and invasion in OSCC 3D-culture models were effectively inhibited by verteporfin, whereas invasion was weakly inhibited by Y27632 and latrunculin. Collectively, the developed OSCC 3D-culture models established with fibroblasts demonstrated the potential for drug screening, with verteporfin showing promising efficacy.