This study aimed to detect the presence of quorum-sensing (QS) signalling molecules and evaluate the in vivo virulence gene expression in shrimp challenged with acute hepatopancreatic necrosis disease (AHPND) positive Vibrio isolates. Three types of QS signal molecules, namely N-acyl-homoserine lactone (AHL), Autoinducer-2 (AI-2) and Cholerae autoinducer-1-like (CAI-1) molecules were detected in AHPND causing isolates using Agrobacterium tumefaciens KYC55, Vibrio campbellii JMH597 and V. campbellii JAF375 biosensors respectively. The presence of QS genes in all the 12 AHPND positive isolates was further justified by conducting molecular screening of the QS-related genes, luxR and luxS. Challenged groups demonstrated a significant (P < 0.05) increase in the expression levels of the QS master regulator gene luxR compared with the unchallenged group. The expression of pirA-, pirB-, toxR- and luxR genes peaked at 36 h in shrimp challenged with AHPND positive Vibrio parahaemolyticus BpShHep31 and V. harveyi BpShHep24. However, compared to the group challenged with V. parahaemolyticus BpShHep31, the V. harveyi BpShHep24 challenged group demonstrated 45.1-, 126.0-, 19.7- and 10.7- fold higher expression of pirA, pirB, toxR and luxR respectively. •The autoinducer-1 (AI-1) and autoinducer-2 (AI-2) are the major quorum sensing signals produced by the AHPND isolates.•Expression level of pirA-, pirB-, luxR- and toxR- regulated virulence genes increased proportionally with high mortality rates in the shrimp challenged with AHPND positive isolates.•Up-regulation of the production capacity of pirA, pirB, toxR and luxR were observed at 36 h.