The bioactive chromium nanoparticles were synthesized by calcination followed by thermal decomposition method. The antibacterial activity of chromium nanoparticles diffused in Dimethyl sulphoxide (DMSO). The antibacterial activity of chromium nanoparticles carried out against significant human pathogens (gram negative bacteria) viz, K. pneumonia, E. coli and P. typhus using agar diffusion cup plate method at 100 µg/ml concentration. The highest zone of inhibition was observed (12.0 mm) against K. pneumonia and lowest zone of inhibition (7.0 mm) E. coli. Thus, the outcomes of these studies suggest that synthesized chromium nanoparticles are of clinical importance.