A 40-day trial was performed to evaluate the effect of short-term natural fresh food (FF) supplementation 20 days prior to ablation in Litopenaeus vannamei broodstock raised under biofloc conditions. Changes in biochemical composition and fatty acids profile were used as indicators of nutritional condition. Females that received FF supplementation (FLOC + FF) achieved better eggs production, spawned more promptly, and presented higher levels of HUFA in eggs as compared to those that did not received FF (FLOC). Proximate analysis of biofloc and microorganisms assessment showed a higher crude protein and lipid content in the FLOC + FF treatment (26.3 and 0.7 %) compared to FLOC treatment (18.4 and 0.3 %) and also demonstrated a higher concentration of filamentous cyanobacteria and nematodes. The better outcomes obtained in females that received short-term FF supplementation justified the FF use in L. vannamei broodstock.