Ternary chalcopyrite CuInSe2 nanoparticles were synthesized by simple wet chemical method. The CuCl2⋅2H2O, InCl3⋅6H2O and Na2SeSO3 were used as the precursor source chemicals for Cu, In and Se ions, respectively. The electron and X-ray diffraction analysis showed the nanoparticles to be near stoichiometric and possess CuInSe2 phase having tetragonal unit cell structure, respectively. The crystallite sizes determined by the Scherrer’s formula (27.67 nm) and Hall–Williamson’s relation (28.32 nm) were in good agreement with each other. The scanning and transmission electron microscopy showed the nanoparticles to be of uniform shape and size. The electron diffraction pattern for selected area of the nanoparticles exhibited ring patterns along with spots stating the nanoparticles to be polycrystalline having well orientated structure within the crystallites. The optical spectroscopy revealed the nanoparticles possess direct optical band gap of 1.9 eV. The Raman spectrum of the nanoparticles showed presence of three peaks. The two peaks positioned at 91 cm−1 and 172 cm−1 are due to CuInSe2 structure, while the third peak at 261 cm−1 is due to CuxSe phase. The anti-microbial study employing the synthesized CuInSe2 nanoparticles showed better anti-microbial effect on Bacillus subtilis compared to Escherichia coli bacteria. The obtained results are discussed in details.