Osteomyelitis remains a serious inflammatory bone disease that affects millions of individuals worldwide and for which there is no effective treatment. Despite scientific evidence that Staphylococcus bacteria are the most common causative species for human bacterial chondronecrosis with osteomyelitis (BCO), much remains to be understood about the underlying virulence mechanisms. Herein, we show increased levels of double-stranded RNA (dsRNA) in infected bone in a Staphylococcus-induced chicken BCO model and in human osteomyelitis samples. Administration of synthetic [poly(I:C)] or genetic (Alu) dsRNA induces human osteoblast cell death. Similarly, infection with Staphylococcus isolated from chicken BCO induces dsRNA accumulation and cell death in human osteoblast cell cultures. Both dsRNA administration and Staphylococcus infection activate NACHT, LRR and PYD domains-containing protein (NLRP)3 inflammasome and increase IL18 and IL1B gene expression in human osteoblasts. Pharmacologic inhibition with Ac-YVAD-cmk of caspase 1, a critical component of the NLRP3 inflammasome, prevents DICER1 dysregulation- and dsRNA-induced osteoblast cell death. NLRP3 inflammasome and its components are also activated in bone from BCO chickens and humans with osteomyelitis, compared with their healthy counterparts. These findings provide a rationale for the use of chicken BCO as a human-relevant spontaneous animal model for osteomyelitis and identify dsRNA as a new treatment target for this debilitating bone pathogenesis.