Recent studies have demonstrated that selenium (Se) and selenium nanoparticles (Se-NPs) exhibited toxicity at a higher concentration. The lethal concentration of Se and Se-NPs was estimated as 5.29 and 3.97 mg/L at 96 h in Pangasius hypophthalmus. However, the effect of different definite concentration of Se (4.5, 5.0, 5.5, and 6.0 mg/L) and Se-NPs (2.5, 3.0, 3.5, and 4.0 mg/L) was decided for acute experiment. Selenium and Se-NPs alter the biochemical attributes such as anti-oxidative status [catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST) activities], neurotransmitter enzyme, cellular metabolic enzymes, stress marker, and histopathology of P. hypophthalmus in a dose- and time-dependent manner. CAT, SOD, and GST were significantly elevated (p < 0.01) when exposed to Se and Se-NPs, and similarly, a neurotransmitter enzyme (acetylcholine esterase (AChE)) was significantly inhibited in a time- and dose-dependent manner. Further, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and malate hydrogenase were noticeably (p < 0.01) affected by Se and Se-NPs from higher concentration to lower concentration. Stress markers such as cortisol and HSP 70 were drastically enhanced by exposure to Se and Se-NPs. All the cellular metabolic and stress marker parameters were elevated which might be due to hyperaccumulation of Se and Se-NPs in the vital organ and target tissues. The histopathology of liver and gill was also altered such as large vacuole, cloudy swelling, focal necrosis, interstitial edema, necrosis in liver, and thickening of primary lamellae epithelium and curling of secondary lamellae due to Se and Se-NP exposure. The study suggested that essential trace element in both forms (inorganic and nano) at higher concentration in acute exposure of Se and Se-NPs led to pronounced deleterious alteration on histopathology and cellular and metabolic activities of P. hypophthalmus.