Candida albicans is among the life-threatening fungal species and the primary contributor to hospital-acquired systemic infections, accounting for nearly 70% of all fungal infections worldwide. The current treatment primarily relies on azoles, pyrimidine analogs, polyenes, and echinocandins. However, growing antifungal resistance highlights the urgent need for the development of alternative treatments against C. albicans. Mesenchymal stem cells (MSCs) offer huge therapeutic potential for the treatment of C. albicans-associated diseases. In this study, palatal adipose tissue-derived MSCs (PAT-MSCs) and PAT-MSCs cultured in 3D biomaterial using nanofibrillar cellulose were tested against C. albicans strains ATCC 10231 and ATCC MYA 2876 using an in vitro antifungal activity assay. In addition, the conditioned medium from both PAT-MSCs and PAT-MSCs cultured in 3D hydrogel biomaterial (CM-PAT-MSCs-3D) were evaluated for their antifungal activities. The combined effect of PAT-MSCs and their secreted factors was also investigated. The expression of five antimicrobial peptide (AMP)-encoding genes was analyzed by quantitative real-time PCR. The expression of antimicrobial peptides was further confirmed via immunocytochemical staining. PAT-MSCs significantly inhibited the growth of C. albicans strains at varying inoculum concentrations (500 and 2000 CFU). Similarly, a comparable antifungal effect was observed when Candida strains were treated with PAT-MSC secreted factors alone. Statistical analysis revealed significant differences between the antifungal activities of PAT-MSCs and CM-PAT-MSCs. Lastly, the combination of PAT-MSCs and CM-PAT-MSC-3D led to a marked reduction in fungal growth, with inhibition rates of 99.75% and 99.91% for C. albicans ATCC 10231 and ATCC MYA-2876, respectively, at 500 CFU inocula. At 2000 CFU inocula, inhibition rates were 99.54% and 99.91%, respectively (****P ≤ 0.0001). These antifungal activities were further confirmed by using RT-PCR and immunocytochemical analysis. Our findings underscore a perspective on the potent antifungal activity of secreted factors from PAT-MSCs cultured within a 3D hydrogel matrix, specifically against various strains of C. albicans. Particularly, the combination of PAT-MSCs with their secreted factors represents a promising therapeutic platform, potentially offering a safer and more effective alternative to conventional antifungal treatments.