The surface microlayer (SML) is the thin biogenic film found at the surface of a water body. The SML is poorly understood but has been shown to be important in biogeochemical cycling and sea-air gas exchange. We sampled the SML of the Blyth estuary at two sites (salinities 21 and 31 psu) using 47mm polycarbonate membranes. DNA was extracted from the SML and corresponding subsurface water (0.4m depth) and microbial (bacteria and archaea) community analysis was performed using denaturing gradient gel electrophoresis of 16S rRNA gene PCR amplicons. The diversity of bacterial functional genes that encode enzyme subunits for methane monooxygenase (pmoA and mmoX) and carbon monoxide dehydrogenase (coxL) was assessed using PCR, clone library construction and restriction fragment length polymorphism (RFLP) analysis. Methanotroph genes were present only in low copy numbers and pmoA was detected only in subsurface samples. Diversity of mmoX genes was low and most of the clone sequences detected were similar to those of mmoX from Methylomonas spp. Interestingly, some sequences detected in the SML were different from those detected in the subsurface. RFLP analysis of coxL clone libraries indicated a high diversity of carbon monoxide (CO)-utilizing bacteria in the estuary. The habitats of the closely related coxL sequences suggest that CO-utilizing bacteria in the estuary are recruited from both marine and freshwater/terrestrial inputs. In contrast, methanotroph recruitment appears to occur solely from freshwater input into the estuary.