Mesenchymal stem cells (MSCs) have emerged as promising alternatives to fight drug-resistant bacterial infections. This study investigates the antibacterial activity of palatal adipose tissue-derived MSCs (PMSCs), particularly when cultured within a 3D nanofibrillar cellulose hydrogel, against four clinically relevant pathogens:K6,ATCC 25,923,K9 andO157:H7. This study showed that both PMSCs alone and PMSCs in 3D cellulose-based hydrogel effectively inhibited the growth of bacterial burden. Notably, PMSCs cultured in the 3D system demonstrated an excellent effect, reducing bacterial burden by up to 14 log inand 12 log inK6 at a 120 µL inoculum after 2 h of incubation. RT-PCR and immunocytochemical analyses found out a remarkable upregulation of the Cathelicidin (LL-37) in PMSCs 3D cultures compared to PMSCs. Furthermore, 3D cellulose-based hydrogel exhibited a significant biofilm-inhibitory effect, reaching a 57.65% reduction. The results demonstrated the importance of 3D cellulose-based hydrogel for treating antibiotic-resistant infections. PMSC therapy based on 3D hydrogel may therefore be offered as more effective antimicrobial agent to overcome drug-resistant bacterial infections.