Background: There is minimal scientific evidence concerning effect of contracted endodontic access cavity design (CEC) combined with Erbium, Chromium: yttrium-scandium-gallium garnet (Er,Cr:YSGG) laser on intracanal bacterial biofilm reduction. Traditional endodontic access cavity preparation (TEC) provides straight-line access to the canal orifices. However, more dentin tooth structure is removed which may affect resistance to tooth fracture under cyclic loading. Aim: To evaluate the effect of access cavity design on the reduction of bacterial biofilm using different disinfection protocols. Materials and Methods: Sound extracted non-restored maxillary first permanent premolars were selected. The samples were divided into two main equal groups according to access cavity design. Each group was subdivided into 3 equal subgroups according to root canal disinfection protocol as following: subgroup A: saline, subgroup B: 5.25% NaOCl,17%EDTA, Subgroup C: Er,Cr: YSSG Laser. Samples were then examined after final disinfection protocol for biofilm bacterial reduction using microbiological culture. Results: Traditional access showed significant higher bacterial reduction than contracted access regarding saline and laser disinfection, while there was no statistically significant difference between both cavity designs using NaOCl. Conclusion: Access cavity design exhibited direct influence on bacterial reduction using Er,Cr: YSGG laser. However, access cavity designs had no influence on bacterial disinfection using NaOCl.