Despite present antiviral agents that can effectively work against HIV-1 replication, side effects and drug resistance have pushed researchers toward novel approaches. In this context, there is a continued focus on discovering new and more effective antiviral compounds, particularly those that have a natural origin. Polysaccharides are known for their numerous bioactivities, including inhibiting HIV-1 infection and replication. In the present study, phosphorylated chitosan oligosaccharides (PCOSs) were evaluated for their anti-HIV-1 potential in vitro. Treatment with PCOSs effectively protected cells from HIV-1-induced lytic effects and suppressed the production of HIV-1 p24 protein. In addition, results show that PCOSs lost their protective effect upon post-infection treatment. According to the results of ELISA, PCOSs notably disrupted the binding of HIV-1 gp120 protein to T cell surface receptor CD4, which is required for HIV-1 entry. Overall, the results point out that PCOSs might prevent HIV-1 infection at the entry stage, possibly via blocking the viral entry through disruption of virus-cell fusion. Nevertheless, the current results only present the potential of PCOSs, and further studies to elucidate its action mechanism in detail are needed to employ phosphorylation of COSs as a method to develop novel antiviral agents.