Aims: The inefficient lysis of recalcitrant bacterial cell wall and subsequent isolation of DNA from environmental samples can lead to a bias in the qualitative and quantitative assessment of bacteria present in the sample. Thus, the selection of an optimum DNA isolation method is the important first step for biosurveillance and metagenomic analyses. This study aims to determine the optimal DNA isolation method out of four commercial DNA isolation kits (A, B, C and D) and two conventional methods (E and F), for rodent faecal droppings. The key selection criterion is the general bacterial diversity contained in the isolated DNA, as evaluated by the Shannon-Weaver index based on the maximal number of PCR-amplicons of partial 16S rRNA gene, derived from each method. The amplicons were separated in accordance with their difference in nucleotide sequences via DGGE. Methodology and results: Five faecal samples of wild rodents were collected from different sites and preserved in DNA/RNA shield reagent (Zymo Research). Each sample was extracted, and the DNA extracts were then subjected to amplification of the bacterial 16S rRNA and DGGE separation of the amplicons. Method E showed a higher yield of DNA (average 324.22 ng/µL) as compared to the other methods. However, the majority of the DNA extracts showed partial degradation. The DGGE profiles showed the highest number of amplicons were generated from DNA extracted from Method A and B with a total of 168 and 167 respectively. This is indicated by the Shannon-Weaver index which were 0.306 and 0.305, respectively. Conclusion, significance and impact of study Method A is the optimum DNA isolation method for rodent faecal samples as its isolated DNA contains the most diverse bacteria. The isolated DNA can then be used for PCR-biosurveillance or metagenomic sequencing and analyses.