High-throughput sequencing of the 16S rRNA gene has considerably helped revealing the essential role of bacteria living on insect cuticles in the ecophysiology and behavior of their hosts. However, our understanding of host-cuticular microbiota feedbacks remains hampered by the difficulties to working with low bacterial DNA quantities as in individual insect cuticle samples, which are more prone to molecular biases and contaminations. Herein, we conducted a methodological benchmark on the cuticular bacterial loads retrieved from two Neotropical ant species of different body size and ecology: Atta cephalotes (~15 mm) and Pseudomyrmex penetrator (~5 mm). We evaluated the richness and composition of the cuticular microbiota, as well as the amount of biases and contamination produced by four DNA extraction protocols. We also addressed how bacterial communities’ characteristics would be affected by the number of individuals or individual body size used for DNA extraction. Most extraction methods yielded similar results in term of bacterial diversity and composition for A. cephalotes (~15 mm). In contrast, greater amounts of artifactual sequences and contaminations, as well as noticeable differences in bacterial communities’ characteristics were observed between the extraction methods for P. penetrator (~5 mm). We also found that large (~15 mm) and small (~5 mm) A. cephalotes individuals harbor different bacterial communities. Our benchmark hence suggests that cuticular microbiota of single insect individuals can be reliably retrieved provided that blank controls, appropriate data cleaning, and standardization of individual body size are considered in the experiment.