Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimu-lation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.