Arthritogenic alphaviruses, such as Ross River virus (RRV) and Barmah Forest virus (BFV), are transmitted by mosquito vectors causing widespread outbreaks of debilitating musculoskeletal manifestations in both the hotter and cooler regions within Australia. Patients experience excruciating pain and inflammation of their joints and surrounding muscle tissues. Current treatments for athritogenic alphaviruses are non-specific and only provide partial relief. A recent study has reported both the involvement of the cartilage structures in RRV pathogenesis and the efficacy of pentosan polysulfate (PPS), a sulfated polysaccharide and a glycosaminoglycan (GAG) mimetic that reduced clinical disease in RRV infected mice. Herein we describe the use of another relevant GAG mimetic to treat arthritogenic infection in vitro. We first assessed compound efficacy through a 50% plaque reduction (IC50) assay. Increasing concentrations of the GAG mimetic compound inhibited plaque formation in viral prototypes; RRV T48, BFV2193, and RRV field isolates, suggesting a strong anti-viral mode of action. To further confirm virus: compound binding, the viral protein compound association constant (KD) was evaluated through isothermal calorimetry titration method (ITC). ITC results confirmed the effective association of virus to the compound. Further, we used human primary chondrocytes and skeletal muscle cells as target cells to establish a productive RRV and BFV infection, which was successfully blocked with the representative GAG mimetic compound. We also report that compound treatment reduced the production of soluble host factors such as IL-6 and MCP-1 at a cellular level. In conclusion, these findings suggest that GAG mimetic therapeutics may have a direct inhibitory effect on athritogenic alphaviral infection and warrant further evaluation for the treatment of other athritogenic alphaviral infections.