Due to limited data on the pathogenicity of Prohemistomum vivax (P. vivax) and its impacts on fish health, this study aimed to determine the morphological, molecular characteristics, pathogenicity, and histopathological alterations in fish infected with P. vivax. Eight hundred (800) Nile tilapia (Oreochromis niloticus) were collected from various farms in Kafr El Sheikh Governorate. The fish were examined for encysted metacercariae (EMC) in different organs. Tissue specimens were collected and underwent histopathological analysis, expression of stress-related genes, and genetic characterization by sequencing of the internal transcribed spacer 2 (ITS2). P. vivax metacercariae were oval to round in shape and were collected from various organs including the muscle, skin, eyes, intestine, liver, kidney, and gills of infected O. niloticus. Sequencing and phylogenetic analysis of the ITS2 region revealed a 507-bp fragment, confirming parasite identity and matching within the same clade as other P. vivax isolates. Infected fish displayed abdominal hydropsy, skin darkening, and emaciation. P. vivax encysted metacercariae were detected during the study period in 620/800 fish, with an overall prevalence of 77.5%. The seasonal prevalence was 95% in summer, 85% in spring, 55% in autumn, and 75% in winter. The intensity of infection was 1–40 cysts per microscopic field. Histopathological examination of muscles revealed parasitic cysts embedded within muscle fibers, causing severe degeneration and necrosis. Upregulation of cytochrome P450 (cpy1a1), heat shock protein 70 (hsp-70), and tumor suppressor p53 (p53) was recorded in both liver and muscle samples of infected tilapia compared to controls. This indicates activation of detoxification, cellular stress, and apoptotic pathways in response to P. vivax infection. There is limited data available on the pathogenicity of P. vivax and its impacts on fish health; thus, this study provides key insights into the morphology, pathogenicity, and histopathological impacts of P. vivax in Nile tilapia.