Cell‐based therapies using adipose‐derived mesenchymal stromal cells (ADMSCs) have shown promising results for the treatment of osteoarthritis (OA). In fact, ADMSCs are now indicated as one of the most powerful cell sources through their immunomodulatory and anti‐inflammatory activities. Recently, an innovative one‐step closed device was developed to obtain microfragmented adipose tissue (MF) to avoid the need for good manufacturing practices for ADMSCs expansion while maintaining their regenerative potential. The aim of this study was to assess the mechanisms of action of MF and ADMSCs from MF (MF‐ADMSCs) on an inflammatory cell model of OA synoviocytes. We found that MF produced low levels of inflammatory factors such as interleukin 6 (IL‐6), CC‐chemokine ligand 5/receptor‐activated normal T‐cell expressed and secreted (CCL5/RANTES), CC‐chemokine ligand 2/monocyte chemoattractant protein‐1 (CCL2/MCP‐1), and CC‐chemokine ligand 3/macrophage inflammatory protein‐1α (CCL3/MIP‐1α), and a higher level only of CXC‐chemokine ligand 8/interleukin 8 compared with MF‐ADMSCs. Matrix metalloproteinase 9 (MMP‐9) degradative factor but released a lower level of its inhibitor tissue inhibitor of the metalloproteinase (TIMP‐1). MF in coculture with synoviocytes significantly induced both the metabolic activity and the release of IL‐6. In contrast, MF, not MF‐ADMSCs, partially decreased CCL5/RANTES. Moreover, MF reduced the release of both macrophage‐specific chemokines (CCL2/MCP‐1 and CCL3/MIP‐1α) and degradative marker MMP‐9. Interestingly, MF increased TIMP‐1 (the MMP‐9 inhibitor) and down‐modulated toll‐like receptor (TLR4) receptor and key molecules of NFκB pathways. These data evidenced different effects of MF versus MF‐ADMSCs on inflamed synoviocytes. MF reduced typical macrophages markers and its potentiality by switching off macrophages activity was strictly dependent on TLR4 and NFκB signaling.