Aims:Thisstudy aimed to assess the risk of reproductive tract contamination in Aceh cattle by Escherichia fergusoniias revealed by 16S rRNA gene sequencing of preputial swab samples.Methodology and results:Preputial swabs taken from 50 breeding bulls at the Indrapuri Breeding and Forage Center of Aceh Cattle, Banda Aceh, Indonesia, were examined for the presence of bacteria. Samples were streaked on MacConkey agar and incubated under aerobic conditions at 37°C for 24 h. Smooth,yellow-or rose-colored colonies were selected for their characteristic appearance andsubjected to further analysis. Genetic identification was based on 16S rRNA gene sequencing and PCR analysis. We conducted a 16S rRNA sequence similarity search with GenBank using BLAST and constructed neighbour-joining dendrogramsusing MEGA. From among closely related species of the genus Enterobacteriaceae, we identified the enteric bacterium E. fergusoniias having the highest sequence similarity.Conclusion, significance and impact of study:We concluded that the E. fergusoniibacterium positively presence in preputial swab samples of clinically healthyAceh cattle population. Accordingly, it is potentially allowing the bacterium to be spread during natural mating or semen collection processing for artificial insemination in cattle breeding farm