This research study is to develop an easy and eco-friendly method for the synthesis of AgNPs using aqueous extract of endophytic fungi, Cladosporium species (CsAgNPs) and investigated the effects of antioxidant, anti-diabetic and anti-acetylcholinesterase (AChE), anti-butyrylcholinesterase (BChE) activity. Cladosporium species-mediated silver nanoparticles were characterized by UV-Vis spectrophotometer, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX). The aqueous extract of Cladosporium species has shown the presence of carbohydrates, tannin, phenolic glycosides, terpenoids, alkaloids, phenol and anthraquinones. At 438 nm conformed the absorbance of AgNPs. The SEM result confirms that size, morphology and high density of the synthesized nanoparticles with huge disparity in the particle size distribution. The FTIR analysis confirmed the important biological compounds responsible for reduction of silver. Strong absorption property of AgNPs was studied by EDX. In antioxidant activity, CsAgNPs showed the involvement of NADPH-dependent reductase in the formation of AgNPs. The AgNPs has reduced the activity of α-amylase, α-glucosidase and dipeptidyl peptidase IV in vitro antidiabetic activity. The CsAgNPs showed significant glucose uptake in 3T3L1 cell line. The AgNPs have shown excellent inhibitory activity against AChE and BChE. To our best knowledge, this is the first on the synthesis of silver nanoparticles using endophytic fungi, Cladosporium species isolated from healthy leaf of Loranthus micranthus. Hence, to validate our results the in vivo animal studies at molecular level are needed to develop an antioxidant, anti-diabetic and anti-cholinesterase agent.