The purpose of this research was to synthesize and characterize gelatin and tamarind gum (TG)/carboxymethyl tamarind (CMT) gum-based phase-separated hydrogels. The hydrogels were thoroughly characterized using bright-field microscope, FTIR spectroscope, differential scanning calorimeter, mechanical tester, and impedance analyzer. The mucoadhesivity, biocompatibility, and swelling property of the hydrogels were also evaluated. The antimicrobial efficiency of ciprofloxacin (model antimicrobial drug) loaded hydrogels was studied against E. coli. The in vitro drug release was carried out in both gastric and intestinal pHs. Microstructural analysis suggested the formation of phase-separated hydrogels. FTIR studies suggested that CMT gum altered the secondary structure of the gelatin molecules. Presence of the polysaccharides within the hydrogels resulted in the increase in the enthalpy and entropy for evaporation of the moisture from the hydrogels. The mechanical studies indicated viscoelastic nature of the hydrogels. Electrical analysis suggested an increase in the impedance of the hydrogels in the presence of the TG. The presence of CMT gum resulted in the decrease in the impedance of the hydrogels. The hydrogels exhibited good mucoadhesivity, biocompatibility, and pH-dependent swelling behavior. The drug-loaded hydrogels showed good antimicrobial activity and the drug release from the hydrogels was pH dependent and diffusion mediated. © 2015 Taylor & Francis.