Selenium nanomaterials (Nano-Se) are new selenium sources with excellent biocompatibility, degradability, and bioactivities. The objective of the present study is the green synthesis of selenium nanoparticles (SeNPs) using Diospyros montana Roxb (DM) bark extract, its characterization, and evaluation for in-vitro antioxidant, antibacterial and anticancer activities. To synthesize Diospyros montana- selenium nanoparticles (DM-SeNPs), selenious acid (H₂SeO₃) was reduced using D. montana extract via precipitation technique. UV-Vis, FTIR, XRD, SEM, EDAX, and ICP-AES were used to characterize DM-SeNPs. The DPPH free radical scavenging assay and reducing power capacity were used to test DM-SeNP for antioxidant activity. The antibacterial properties of the DM-SeNP were tested using the well diffusion method against gram-positive and gram-negative microorganisms. DM-SeNPs were also subjected to antiproliferative activity using MTT assay via MCF-7 cell line. A peak in UV at 289 nm validated the synthesis of DM-SeNPs. According to DLS, SEM, and TEM images, the size of DM-SeNPs was between 100-150 nm. XRD analysis confirmed the crystallinity of DM-SeNPs. Selenium was verified in colloidal dispersion using EDAX analysis, and ICP-AES confirmed selenium content 63.45 ±18.3 µg/mL in DM-SeNP. The IC₅₀ 24.72 ± 0.63 µg/mL and EC₅₀ 46.30 ± 0.21 µg/mL values indicated that the DM-SeNPs had a good antioxidant capacity. DM-SeNPs showed comparative better antibacterial potential. The inhibition zones were found to be the highest for E. coli (48.00 mm), B. subtilis (44.14 mm), Klebsiella pneumonia (36.20 mm), and S. aureus (34.16mm), respectively. Antiproliferative activity was carried out, which showed DM-SeNPs were cytotoxic to breast cancer cells line (MCF-7). The IC₅₀ values for DM-SeNPs were found to be 38.19 ± 0.27 µg/mL and Doxorubicin 6.41 ± 0.09 µg/mL, respectively. The study suggests that DM-SeNPs display moderate cytotoxicity that could dose-dependently inhibit cell proliferation. Thus, experimental evidence provides insight into selenium nanoparticle synthesis, its potential therapeutic value, and the prospect of developing a formulation containing DM-SeNPs.