Ecytonucleospora hepatopenaei (EHP), a microsporidian parasite first named and characterized from the Penaeus monodon (black or giant tiger shrimp), causes growth retardation and poses a significant threat to shrimp farming. We observed shrimp farms associated with disease conditions during our fish disease surveillance and health management program in West Bengal, India. Shrimp exhibited growth retardation and increased size variability, particularly in advanced stages, exhibiting soft shells, lethargy, reduced feeding and empty midguts. Floating white feces were observed on the surface of the pond water. Suspecting a microbial infection, the shrimp samples were collected and aseptically brought to the ICAR-CIFRI laboratory for molecular confirmation. A nested PCR was used to screen shrimp tissue, feces, feed and environmental samples for the possible presence of hepatopancreatic microsporidiosis caused by Ecytonucleospora hepatopenaei. The results confirmed that the shrimp samples were positive for EHP. Histopathological investigation revealed mature spores in the HP tubule lumen and epithelial cells along with necrotic tubule in the symptomatic group. Further, the transcription analysis revealed that ProPO, Hsp70 and α2-macroglobulin genes were significantly upregulated, while decreased expression of LGBP, PXN and Integrin ß was observed in shrimp infected with Hepatopancreatic microsporidiosis. Furthermore, compared with the healthy group, significant intestinal bacteria changes were observed in the EHP-infected group. The in vivo survival assay, using crustacean animal model Artemia franciscana, suggests that symptomatic shrimp gut samples harbour pathogenic Vibrio parahaemolyticus, V. harveyi and V. campbellii. These results significantly advance our understanding of the molecular and ecological aspects of EHP pathobiology.