A range of sampling methods are available to sample the sea surface microlayer, including mesh screens (MS), glass plates (GP), hydrophobic (PTFE, polytetrafluoroethylene) and hydrophilic (PC, polycarbonate) membranes. We compared bacterial community structures in surface microlayer samples and in subsurface water (0.4 m) collected using the above sampling methods. Samples were collected from the Tyne Estuary and analysed using DGGE of bacterial 16S rRNA genes. Samples collected using GP and MS were similar to subsurface water samples. Samples collected using PTFE and PC showed distinct differences compared to samples collected using GP and MS and subsurface samples. Membranes sample within the proposed depth range of the actual sea surface microlayer while GP and MS collect deeper samples which therefore also contain subsurface water. There is therefore clear evidence that GP and MS samplers under-represent the surface microlayer. Bacterial and archaeal communities were compared in sea surface microlayer and subsurface samples collected with PC from 3 sites in the Pacific Ocean. Samples were analysed using DGGE of bacterial and archaeal 16S rRNA genes. Bacterial communities in samples collected from the sea surface microlayer at 2 sites on either side of Oahu Island were more similar to each other than to their cognate subsurface samples. Archaeal communities did not show a distinct surface microlayer effect. Data presented here clearly indicate that Bacteria respond significantly to this sea surface microlayer environment and further support the use of PC in sampling surface microlayers.