Background At present, research on facile, green synthesis of nanoparticles has significantly increased because of its fast, one-step, cost-effective, time-efficient, and non-toxic nature. In this study, we have reported a single-step green synthesis of copper nanoparticles using cell wall polysaccharides of a hot spring origin, thermotolerant Bacillus species. Result Copper nanoparticles were characterized using UV-visible spectrophotometry, fluorescence and Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive spectroscopy, particle size, and zeta potential analyses. UV-visible spectra of synthesized copper nanoparticles exhibited a band cantered between 220–235 nm, characteristic spectra of copper oxide nanoparticles. Infrared spectra showed the band at 490-530 cm−1 corresponding to metal-oxygen or copper nanoparticle vibration, supporting the presence of copper oxide nanoparticles in the monoclinic phase. The energy dispersive spectra of copper nanoparticles exhibited a strong signal from elemental copper. The dynamic Light Scattering pattern confirmed the nanoparticle nature of the studied sample. These nanoparticles showed preferential activity against gram-negative pathogens, Salmonella typhi and Escherichia coli. The thermodynamic nature of the nanoparticles is also established for its antibacterial actions. Conclusions The antibacterial action and its thermodynamics reinforce the possible use of copper nanoparticles as an alternative to commercially available antimicrobials. This study may open a new path for future studies to treat harmful microorganisms resistant to traditional antibiotics in a greener way.