The aim of the present investigation is to assess the sublethal toxicity of biologically synthesized silver nanoparticles (Ag NPs) in Indian major carp Labeo rohita. Ag NPs used in the study were synthesized by using AgNO3 with aqueous leaf extract of Piper nigrum. Median lethal concentration (LC50) of synthesized Ag NPs was determined for 96 h (25 μg/L); 2.5 μg/L (1/10th LC50) and 5 μg/L (1/5th LC50) were taken as sublethal concentrations to evaluate the toxicity for 35 days. The results of the TEM, SEM, and EDX analyses revealed that Ag NPs were considerably accumulated in the gill, liver, and kidney of fish at both concentrations (2.5 and 5 μg/L). Consequently, the activity of the antioxidant enzymes, SOD and CAT, was significantly (P < 0.05) decreased in the gill, liver, and kidney when compared to the control group during the study period. However, lipid peroxidase (LPO) activity in the gill, liver, and kidney was significantly (P < 0.05) increased, and the result concluded a possible sign of free radical-induced oxidative stress in Ag NP-exposed fish than the sham-exposed individuals. The histopathological study also confirmed the alterations such as degeneration of lamella, lifting of lamellar epithelium, hepatic necrosis, pyknotic nuclei, increased intracellular space, and shrinkage of glomerulus elicited by Ag NPs in the gill, liver, and kidney of Labeo rohita with two different concentrations. The findings of the present study revealed that green synthesis of Ag NPs from Piper nigrum at sublethal concentrations leads to accumulation of Ag, oxidative stress, and histopathological alterations in the target organs of the fish, Labeo rohita.