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The main challenge in shrimp farming is the high prevalence of diseases caused by pathogenic bacteria from the Vibrionaceae family. Natural extracts enriched with prebiotic and antibacterial compounds have been proposed as alternatives to the controversial use of antibiotics. In this study, a flavonoid-enriched extract (11.54 ± 1.81 mg QE/g DW) obtained from Agave lechuguilla bagasse was evaluated as a feed supplement at 0.0, 0.1, 0.3, and 0.6% in the diet for the Pacific white shrimp (Penaeus vannamei), before and after infection with Vibrio parahaemolyticus strain CIBGEN-003. The effect of the lechuguilla extract on the gut microbiome and vibriosis prevalence was assessed through high-throughput sequencing of 16S rRNA genes analysis, comparing the bacterial community at 0 h, 48 h, and 72 h of the challenge with the pathogenic strain. A global shift in the relative abundance was observed at phylum and class level and among the dietary treatments during the infection. In particular, an increase in the abundance of Vibrionaceae was observed. A higher survival of the shrimp fed with the lechuguilla extract was observed compared to control, and a lower vibriosis prevalence with the two highest inclusion levels, 0.3 and 0.6%. These findings were related to the differences in the microbial community, according to the inclusion level of the lechuguilla extract before the inoculation of V. parahaemolyticus. Results provide a new perspective to further understand the required structure of the gut microbiome to prevent diseases in shrimp farming. Modulation of the gut microbial community by the flavonoids, quantified in the lechuguilla extract, significantly increased the resistance of P. vanamei against V. parahaemolyticus, supporting the use of plant extracts as alternatives to traditional disease-fighting aquaculture practices. • The flavonoid-enriched extract, used in this study, contain flavonoids known as a promoters of microbial diversity and anti-inflamatory effects. • This is the first time that a flavonoid-enriched extract from Lechuguilla is applied as fed supplement for shrimp. • The shrimps fed with the diets included with the Lechuguilla extract presented a diferent microbial structure than the control. • The addition of 0.3% and 0.6% of the Lechuguilla extract as fed additive gave resistance to the animals against vibriosis. [ABSTRACT FROM AUTHOR]