Susceptibility to antimicrobial agents among Enterobacteriaceae recovered from feces of the Okinawa least horseshoe bat Rhinolophus pumilus (OLHB) in Japan was investigated in 78 isolates. Of these, one isolate was resistant to chlortetracycline and streptomycin, and nine were resistant to sulfadimethoxine (SDMX). Half of these (n = 5) resistant isolates had the transmissible R plasmid for antimicrobials suggesting that OLHB is a species that hardly harbors antimicrobial-resistant Enterobacteriaceae. One possible reason for this low prevalence of antimicrobial resistance is that the bat lives in a clean environment with no or only very low artificial drug contaminations due to low contact with such as livestock that frequently carries antibiotic-resistant bacteria. This study suggests that it's important to monitor antimicrobial resistance of Enterobacteriaceae in OLHB as a model for natural R plasmid transfer in a natural environment without specific pathological bacteria and artificial drugs.