The study was designed to compare the extent of bacterial colonisation on the surface of Polylactic acid/Polyglycolic acid copolymer and expanded Polytetrafluoroethylene based Guided tissue regeneration (GTR) membrane in an in vitro model by spot analysis and scanning electron microscopy. Earlier in vitro models have aimed to study the barrier function against the bacterial penetration across just one surface of the GTR membranes. No such study is present in the literature which aimed to quantify bacterial adhesion over both the surfaces of the membrane. Sterile Resorbable PLA/PGA copolymer and non-resorbable microporous ePTFE based GTR membrane were used in the study. Both groups were subdivided into two subgroup (n=10) based on incubation period of 24 and 48 hours. Incubated in Todd Hewitt broth with Streptococcus mutans, the samples were vortexed and analysed for bacterial count using spot test and scanning electron microscopy. Between PLA/PGA and ePTFE GTR membrane at 24 hours incubation time period, there was a significant difference in terms of Colony forming units (p = <0.001), with the median Colony forming units being highest in the PLA/PGA GTR membrane. At 48 hours incubation time period, there was a significant difference in terms of Colony forming units (p = <0.001), with the median Colony forming units being highest in the PLA/PGA GTR membrane. Our findings depict that ePTFE based GTR membrane showed significantly lesser bacterial colonisation on its surfaces as compared to PLA/PGA at both the incubation periods i.e., 24 hours and 48 hours as shown by both spot test and SEM.