Green fabrication of nanoparticles (NPs) using biological sources is the fast-growing trend replacing chemical synthesis via toxic materials. Considering the importance and feasibility of green fabricated NPs, the present research focuses on the synthesis of gold nanoparticles (AuNPs) using the aqueous extract of the endophytic Cladosporium sp. (MycoAuNPs) isolated from Commiphora wightii. The synthesized MycoAuNPs are characterized using UV-Vis spectroscopic, FTIR, X-ray diffraction (XRD) analysis, and transmission electron microscopy (TEM). The synthesized NPs showed a sharp absorption peak at 524 nm, with an average size between 5 and 10 nm in a spherical shape. XRD revealed the crystalline nature, and EDX profiling confirmed the presence of gold (Au) and oxygen (O) atoms. The biological potential of MycoAuNPs were tested under both in vitro and in vivo conditions. MycoAuNPs showed anti-cancer activity in breast cancer cell line MCF-7 (IC 38.23 µg/mL) through the induction of apoptosis. Further, MycoAuNPs showed potential against growth of tumor in tumor-bearing mice models. MycoAuNPs significantly reduced the body weight, ascites volume, and increased the lifespan of EAC bearing mice. It induced apoptosis of the EAC cells, which was confirmed by DNA fragmentation and Giemsa staining. Also, they did not develop any secondary complications or side effects in normal mice. The photocatalytic activity of MycoAuNPs tested against Rhodamine B and Methylene Blue dyes showed potential dye degradation in the presence of sunlight. Thus, the present study gives a clear idea of the multifaceted therapeutic and catalytic applications of the biosynthesized MycoAuNPs.