Osteoarthritis is a whole-joint disease. While some intra-articular tissues are in physical contact with each other, it is the synovial fluid that acts as a major connecting medium into which joint tissues and cells release their bioactive molecular content. Osteoarthritic synovial fluid contains a plethora of systemic and locally derived biomolecular factors, including cells, extracellular vesicles, proteins, crystals, metabolites, and RNAs. While many of these biomolecular factors are primarily considered as potential biomarkers for OA diagnostics, the bioactivity relayed by these factors and their critical contributions to osteoarthritis pathobiology have received less attention. In this review, we highlight insights into the bioactivity of molecular constituents contained within human osteoarthritic synovial fluid, its intrinsic bioactivity, as well as its potential, and the barriers to use synovial fluid to biomolecularly stratify individuals for specific targeted therapies or osteoarthritis stage.