Stool collection devices minimizing the exposure of gut bacteria to oxygen are critical for the standardization of further microbiota-based studies, analysis and developments. The aim of this work was to evidence that keeping anaerobiosis has a deep impact on the viability and diversity of the fecal microbiota that is recovered in the laboratory. Recovering certain microbial populations, such as obligate anaerobic bacteria, is particularly critical if the purpose of the study is to envisage personalized therapeutic purposes, such as autologous Fecal Microbiota Transplant. In this study the same fecal specimens were sampled in conventional stool containers and GutAlive, a disposable device that minimizes exposure of the gut microbiota to oxygen. Samples from five healthy donors were analysed and 150 differential colonies were recovered and identified by 16S rRNA gene sequencing. Globally, GutAlive maintained extremely oxygen sensitive (EOS) populations that were lost in conventional stool containers, and thus viability of species such as as Akkermansia muciniphila, Faecalibacterium prausnitzii and a novel member of the Clostridiales order was kept. These obligate anaerobes were not recovered using the conventional stool collection device. In conclusion, the use of GutAlive for stool collection and transport optimized the viability and recovery of EOS bacteria in the lab by diminishing oxygen toxicity.