Biosynthesized nanoparticles are gaining attention because of biologically active plant secondary metabolites that help in green synthesis and their unique biological applications. This study reports an ecofriendly, reliable, and cost-effective synthesis of silver nanoparticles using the fruit extract of Citrus maxima (C. maxima). The biosynthesized silver nanoparticles (AgNPs) were characterized using UV–visible spectroscopy and transmission electron microscope. To determine in vivo cytotoxicity of biosynthesized AgNPs and to compare the effects of different AgNP doses. On rats, we conducted an in vivo. The rats were given three dosages of AgNPs (5mg, 7mg, and 10 mg), and three-month-old rats were placed into five groups, each with six rats. A liver function test (ALT, AST, Bilirubin, Alkaline phosphate) and histological parameters were performed as preventive measures. The toxic effect of AgNPs on rats was dose- and time-dependent, where no side effects or lethality was observed in the tested animals over the study period. Our data showed that AgNPs at low dosages (5 and 7 mg/kg) were acceptable for biomedical usage and had no negative effect. In contrast, higher doses (10mg/kg) may be cytotoxic or lethal. Our study shows that high doses of AgNPs can be lethal to eukaryotic cell-based systems. In addition, we have also discovered that azithromycin was more hazardous than AgNPs. This further suggests the necessity of comprehensive in vivo studies to determine the extent of nanotoxicity.