The current study evaluated the effects of genistein (GEN) supplementation to alleviate the arsenic (As)-induced hepatotoxicity in Oreochromis niloticus. This was conducted in two steps: a computational prediction study (in silico) and an experimental investigation (in vivo). The prediction step involved molecular docking analysis to assess the interactions between GEN and key stress-related mRNAs in Nile tilapia. In the experimental phase, 160 Nile tilapia fingerlings were randomly assigned to four treatment groups (in four replicates/group) for 60 days: (1) a control group fed a basal diet, (2) a GEN group receiving a GEN-supplemented diet (500 mg/kg), (3) an As group exposed to 10 µg/L As, and (4) an As + GEN group, in which fish was exposed to As and fed the GEN-supplemented diet. The computational assessment of GEN’s binding ability revealed strong interactions with key mRNAs associated with inflammation and misfolded protein responses. The in vivo results revealed that GEN significantly alleviated As-induced hepatic oxidative stress and hepatocellular damage by restoring liver enzyme levels, lipid profiles, and bilirubin content and restoring the serum proteins to near-normal values. Additionally, GEN downregulated the expression of endoplasmic reticulum (ER) stress- and inflammation-related genes in the liver tissue of the As + GEN group, compared to the As-exposed fish fed on a basal diet. Additionally, the histopathological analysis further confirmed that GEN supplementation mitigated hepatic tissue damage, reducing necrosis, congestion, and inflammatory cell infiltration. In conclusion, GEN supplementation effectively counteracted As-induced hepatotoxicity in Nile tilapia by modulating oxidative stress, ER stress, and inflammation while preserving liver structure and function. Also, the molecular docking results suggest that GEN interacts with the mRNAs of inflammatory and misfolded protein targets, which are increased due to exposure to As-contaminated water. All our findings highlight GEN as a promising natural dietary additive for improving hepatic health in fish inhabiting As-contaminated environments.