White spot syndrome virus (WSSV) is one of the most virulent pathogens afflicting shrimp farming. Understanding its influence on shrimp intestinal microbiota is paramount for the advancement of aquaculture, since gut dysbiosis can negatively impact shrimp development, physiology, and immunological response. Thereupon, the data presented herein assesses the influence of WSSV infection and different rearing systems on the intestinal microbiota of Penaeus vannamei. Our study aimed to describe and correlate the composition of shrimp (Penaeus vannamei) gut microbiota, when reared in biofloc and clear seawater, before and (48 h) after WSSV experimental infection. Shrimp were kept in two different systems (biofloc and clear seawater) and experimentally infected with WSSV. Intestine and water samples were characterized by 16S rRNA gene sequencing, before and after viral infection. We observed (i) WSSV induced higher mortality among shrimp reared in biofloc; (ii) WSSV led to a loss of intestinal microbiota heterogeneity, at the genus level, in shrimp kept in clear seawater; (iii) there was a prevalence of Cetobacterium and Bacillus in the intestine of shrimp from both systems; (iv) WSSV did not cause significant changes in intestinal microbiota diversity or richness; (v) regardless of the type of system and time of infection, intestinal microbiota was dissimilar to that of the surrounding water, despite being influenced by the type of system. Therefore, WSSV infection leads to punctual dysbiotic changes in shrimp microbiota, although the virus is sufficiently virulent to cause high mortalities even in well-managed systems, such as a balanced experimental biofloc system. KEY POINTS: • WSSV infection leads to a perturbed gut microbiota in shrimp. • WSSV infection greater impacts microbiota of shrimp reared in CSW than those in BFT. • WSSV infection caused higher mortality levels in shrimp reared in BFT than in CSW. • Rearing system influences shrimp gut microbiota composition. Graphical abstract.