Abstract Background Effective means are needed to efficiently collect fecal samples for microbiome analysis in large-scale epidemiological studies. Using twenty-four fecal aliquots prepared from three healthy individuals, we compared the following four fecal sample collection methods for assessment of human gut microbiome: 1) fecal occult blood test cards, held at room temperature for three days, 2) Eppendorf tubes, at room temperature for three days, 3) Eppendorf tubes with RNAlater, at room temperature, and 4) as controls, samples immediately frozen at −80°C. The 24 samples were assayed by 16S rRNA gene sequencing to compare overall microbiome structure and taxon distributions according to collection method. Results Storing fecal occult blood test card samples at room temperature for three days did not affect total DNA purity and relative 16S rRNA bacterial gene contents, compared with fresh frozen collection. Overall microbiome structure, based on phylogenetic UniFrac index, differed significantly by subject (p = 0.001), but microbiome structure (p = 0.497) and relative abundance of major microbial taxa (phyla) (p > 0.05) did not differ significantly by collection method. Conclusions Our findings suggest that low-cost fecal occult blood test card collection may be a feasible means of sample collection for fecal microbiome assessment in large-scale population-based studies.