Objectives: This research was aimed to evaluate a novel approach for preparation of mucoadhesive microspheres which can reside in the gastrointestinal tract for an extended time period. The microspheres contained amoxicillin, an anti-bacterial agent useful for the eradication of Helicobacter pylori. Methods: Ten different formulations were prepared by chemical cross-linking technique using gluteraldehyde as a cross linking agent and chitosan as mucoadhesive polymer. Natural release retardant polymers like guar gum, gum ghatti and xanthan gum were employed. All the microspheres were characterized for morphology, particle size, drug entrapment efficiency, swelling index, bioadhesion to mucosal tissue and in vitro drug dissolution and anti-bacterial activity against E. coli. Results: The FTIR and DSC data indicated that there were no interactions between the drug and polymers used. All the microspheres exhibited good flow properties. The microspheres had a spherical shape with rough surface. The microspheres showed a good mucoadhesivity and also anti-bacterial activity. The release of the drug was prolonged to 12h when incorporated into mucoadhesive microspheres. Conclusion: Data obtained in this study concluded that mucoadhesive microspheres of amoxicillin can be used to effectively clear H. pylori from the gastrointestinal tract due to prolonged residence time resulting from mucoadhesion. In this study drug release was diffusion controlled and followed zero order kinetics.