Background Ocimum sanctum (OS) is a medicinal plant with antioxidant, anti-cancer, anti-diabetic, antimicrobial, and anti-inflammatory activities. Extracellular matrix (ECM) maintains the structural stability of tissues. Hyaluronic acid (HA), which is used in hydrogel fabrication and osteochondral regeneration, increases cell viability and the expression of marker genes. Periodontal ligament stem cells (PDLSC), which are a type of mesenchymal stem cells (MSC), have self-renewing capacity and they prevent teratoma formationand promote tendon (TEN) regeneration. The aim of this study is to incorporate the phytochemical effects of Ocimum sanctuminto hyaluronic acid hydrogel scaffolds made with the MSCs in tendon ECM for increased tissue regeneration. Materials & methods Ocimum sanctum extract and methacrylated hyaluronic acid (HA-MA) were prepared. An ovine tendon sample was decellularised to obtain the ECM. The study groups of HA, TEN, HA_OS, HA_TEN, and HA_OS_TEN were prepared. The presence of tendon cells was confirmed by picrosirius red staining and the hydrogel scaffolds were analysed using scanning electron microscopy (SEM), swelling, differentiation, compression, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) compatibility analyses. Results The morphology of the samples was analysed by SEM analysis. The HA_OS_TEN sample showed the highest rate of tenogenesis, lowest swelling, high cell viability and differentiation, and optimal compression rates. Conclusion This study showed that hyaluronic acid combined with Ocimum sanctumand tendon ECM is a very good conjugation for the preparation of hydrogel scaffolds for tendon tissue regeneration using MSCs.