Introduction: Laryngectomy patients undergo voice rehabilitation that requires implantation of trachea-oesophagal speech valves (TESV). Usually, laryngeal cancer patients require insertion of these devices post-operatively to im-prove their quality of life. Implantation of TESV dates back to 1979 by pioneering work of Blom and Singer. There are cases of aspiration of TESV wearer reported, and obstruction of the TESV causes leakage through the valve and is suggested as a main reason for replacement of the device. The dysfunctional failure may be caused by microbial colonization on the valve or physical malfunction and requires immediate replacement is desirable. The aim of this study is to identify the microbial community members of selected TESVs using both culture-independent techniques (Next-generation sequencing) to analyse the microbiota, including unculturable species, and routine microbiology techniques (culture-dependent method) and to obtain representative isolates that can form the basis for experiments to enable increased understanding of the community. Methods: Biofilms were harvested from 16 explanted speech valves from patients visiting the ENT clinic in Freeman Hospital, Newcastle, UK. Routine microbiology techniques (culture-dependent method) including ChromeID® plates and Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometry were used for identification of TESV microbiome. Sequencing of the samples was performed at MR DNA (www.mrdnalab.com, USA) on a MiSeq following the manufacturer’s guidelines in order to determine the bacteria and candida composition in the biofilm community. Results: The most frequently isolated fungal species was C. albicans, which was cultured from 11 out of 16 TESVs (79%), followed by five TESVs with C. tropicalis (36%), three TESVs had C. glabrata (21%) and only one TESV contained S. cerevisiae (7%). Interestingly no biofilm communities contained more than two fungal species and 2 TESVs (12%) possessed only bacterial species. There were only 16 species of bacteria cultured and identified by MALDI-TOF MS. This was far lower than the 91 species that were detected by NGS. Species from the genus Lactobacillus were found in 10 of 16 TESVs (63%), the highest frequency of any bacterial genus isolated from TESVs followed by S. aureus found in eight TESVs of 16. S. epidermidis was identified in two TESVs (13%), Streptococcus spp., K. oxytoca and O. anthropi were both identified in five different TESVs, while the gut bacterium E. faecium was found in four TESVs. Only one TESV contained E. coli. Conclusion: TESV biofilm composition was dominated by Candida spp. and occasionally contained other types of eukaryote such as Saccharomycetes. It was not uncommon for more than one Candida species to be present. The biofilms also harboured a mixture of bacteria, with lactic acid producers (Lactobacillus sp. and Streptococcus sp.) normally accompanying Candida sp. in the biofilm.