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The impact of grapefruit seed extract (GFSE) as an antibacterial agent on citric acid (CA) crosslinked sodium carboxymethylcellulose (NaCMC)/hydroxypropylmethylcellulose (HPMC) hydrogel films has been studied by incorporating different quantities of GFSE. The prepared films were examined for their physical, thermal, mechanical and antibacterial properties. It was observed that crystallinity and initial decomposition temperature of hydrogel films decreased with GFSE concentration. Furthermore, the swelling degree and tensile strength of hydrogel films were found to be 257.29 ± 5.08%–162.06 ± 1.78% and 11.61 ± 0.27–2.21 ± 0.94 MPa for increasing GFSE concentration varying from 0.25% - 1.5% (v/v). The presence of nanoparticles in the films was observed by FESEM and FETEM analysis. It was confirmed that the formation of nanoparticles (micelles) is due to the addition of NaCMC and GFSE, probably glycerides, which is one of the main components in GFSE. The hydrogel films have demonstrated excellent antimicrobial activity and elongation at break (%). Moreover, zeta potential of nanoparticles was recorded to be −55.26 mV ascertaining their stability in water that contributed to a higher antimicrobial activity against gram negative bacteria. All these outcomes prove the nanocomposite films to be a potential substitute for hydrogels loaded with synthetic drugs in wound healing and other biological applications.