In this study, the endophytic fungus Coniothyrium chaingmaiense-KUMBMDBT-25 was isolated from the healthy stem of Euphorbia tirucalli, mass cultivated by submerged fermentation, and extracted using ethyl acetate as a solvent. The extract was subjected to GC-MS analysis. The synthesized Con-AgNPs were characterized through various bioanalytical methods. The synthesis was confirmed by Bio- spectrophotometry, which showed an absorption peak at 404 nm. FTIR analysis verified the reduction and capping of Con-AgNPs, displaying peaks corresponding to various functional groups. SEM-EDAX and HR-TEM examinations revealed that the Con-AgNPs were spherical, and EDAX analysis confirmed the presence of silver atoms at 3 keV. XRD studies revealed the crystalline structure of Con-AgNPs. DLS and Zeta potential tests determined the size and stability of the synthesized Con-AgNPs, which were 65.81 nm. The Con-AgNPs demonstrated strong antibacterial activity against P. aeruginosa (14.06 ± 0.11 mm, 10 mg/mL) and effective antifungal activity against A. flavus (13.03 ± 0.05 mm, 10 mg/mL). Con-AgNPs exhibited notable biological attributes, including a cytotoxic effect of up to 38.82% and 19.15% at 200 µg/mL in an MTT assay measuring cell viability. Additionally, the nanoparticles demonstrated significant anti-inflammatory effects in both in vitro and in vivo studies, validating the biological and pharmacological potential of Con-AgNPs.