Cystoseira usneoides is a brown algae commonly found in the Portuguese coast. A previous study has reported high antioxidant activity of its extract, however, few works have been made in order to isolate and identify the molecules responsible for the antioxidant activity. In this work, a vacuum liquid chromatography (VLC) was performed to Cystoseira usneoides dichloromethane extract using silica gel with the following solvents system: 100% cyclohexane followed by a mixture of 3:1; 1:1; 1:3 of cyclohexane and ethyl acetate and 100 % ethyl acetate. The five fractions were concentrated in a rotary evaporator and dissolved in DMSO at 100mg/ml. The antioxidant potential of all fractions was evaluated by both, the quantification of the total phenolic content (TPC) and by the evaluation of the DPPH radical scavenging activity. Comparatively to the initial values of DPPH radical scavenging activity (IC50= 81.8 µg/ml (54.74-122.2)) and the total phenolic content (86.28 ± 0.002 mg of gallic acid eq./g of extract) of the crude dichloromethane extract, all the fractions obtained by VLC in a silica gel column reported much less antioxidant activity. The most active fractions scavenging the DPPH radical were F3, F4 and F5 which presented and IC50 of 1314 (1209-1429), 2084 (1624-2674) and 1414 (1199-1668) µg/ml, respectively. F5 fraction presented the highest phenolic content of 20.85 mg of gallic acid equivalents/g of extract. The results suggest that the compounds we are trying to isolate have strong affinity with the stationary phase, and therefore, weren’t eluted with the solvents, even with the increasing polarity along the chromatography. Even though VLC, with a silica gel column, have already revealed to be efficient for separation of compounds extracted with dichloromethane, as regards to antioxidant compounds, these continue to be a great challenge.