The present study was aimed at evaluating the fluorescence property, sporicidal potency against Bacillus and Clostridium endospores, and surface disinfecting ability of biogenic nano silver. The nano silver was synthesized using an actinobacterial cell-filtrate. The fluorescence property as well as imaging facilitator potency of this nano silver was verified adopting spectrofluorometer along with fluorescent and confocal laser scanning microscope wherein strong emission and bright green fluorescence, respectively, on the entire spore surface was observed. Subsequently, the endospores of B. subtilis, B. cereus, B. amyloliquefaciens, C. perfringens and C. difficile were treated with physical sporicides, chemical sporicides and nano silver, in which the nano silver brought about pronounced inhibition even at a very low concentration. Finally, the environmental surface-sanitizing potency of nano silver was investigated adopting cage co-contamination assay, wherein vital organs of mice exposed to the nano silver-treated cage did not show any signs of pathological lesions, thus signifying the ability of nano silver to completely disinfect the spore or reduce the count required for infection. Taken these observations together, we have shown the multi-functional biological properties of the nano silver, synthesized using an actinobacterial cell-filtrate, which could be of application in advanced diagnostics, biomedical engineering and therapeutics in the near future.