Aims There has been growing interest in faecal microbiota transplantation (FMT) as treatment. Although, frozen donor faeces preserved at -20 degrees C has been widely used for practical advantages, freezing at -20 degrees C can affect bacterial viability. Adequacy evaluation of fresh and frozen faeces as the transplant is necessary for the methodological improvement of FMT. Methods and Results The viable bacterial compositions of faecal specimens under fresh and freezing conditions were compared by a microbiome analysis using propidium monoazide (PMA microbiome). In addition, recovery abilities from bacterial reduction by antibiotics were compared between fresh and frozen FMT using a murine model. PMA microbiome results suggested that freezing and freeze-thawing did not significantly affect in vitro faecal bacterial viability. However, the recovery effect from antimicrobial cleansing in frozen FMT was reduced in a freezing time-dependent manner, especially prominent in Actinobacteria and Bacteroidetes phyla. Conclusions Short-term freezing preservation of faeces exhibited maintenance of enteric colonization ability in frozen FMT in comparison to 1 month -20 degrees C-preservation. Significance and Impact of the Study: Long-term similar to 20 degrees C-preservation of transplanted faeces can result in instability of the clinical outcome in FMT therapy. The standardization of practical procedures of FMT therapy according to disease types is desirable.