First generation drugs such as zidovudine have been extensively used in clinical practice, resulting in the development of HIV resistance to these nucleoside analogs. Several studies have demonstrated the effective anti-HIV activity of natural products derived from seaweeds, suggesting promising sources of substances for the development of novel antiviral drugs. In this paper, the antiviral effect of extracts from the red seaweed Acanthophora spicifera on HIV-1 replication was evaluated in vitro. Peripheral blood mononuclear cells obtained using the Ficoll-Hypaque gradient were used for cytotoxicity and antiviral activity testing. The dichloromethane extracts, ethyl acetate, acetone, and methanol were found to have CC50 values of 31±7.4, 45±11, 38±3.5, and 179±25 μg/mL, respectively. With the control, the extract prepared in ethyl acetate inhibited approximately 60% of the viral load, which is the best result among the extracts. This same extract showed an IC50 value of 33.17±4.84 μg/mL for the reverse transcriptase. The EtOAc extract from A. spicifera showed to be an efficient HIV antiviral due to its phenolic compounds, as evaluated by nuclear magnetic resonance.