The antibiotic resistance has become a major public health problem globally. Combinatorial administration of natural compounds and antibiotics is an alternative approach, particularly if synergistic effects can be elicited against multi-drug resistant bacteria. The aim of this study was to investigate the combinatorial activity of propolis and gentamycin against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 in three dimensional (3D) thermo-sensitive hydrogel (Poloxamer 407 25% (w/w)) to mimic the microenvironment realistically. Rheological properties, sol-gel transition temperature and the porosity of the hydrogel were determined. Subsequently, thermo-sensitive gel was loaded with propolis and gentamycin for evaluation of the combined effect. The fractional inhibitory concentration indices showed marked synergy and partial synergy without and within 3D termo-sensitive hydrogel at a range of 0.38 to 0.56, respectively. This 3D platform can be utilized to screen potential antimicrobial compounds and readily expanded to other microbial species.