DNA extraction is a fundamentally important step for the implementation of genotypic techniques in microbial identification, and the use of such techniques has become essential for the analysis of soil microbial diversity. Considering culture independent methodologies, it is still necessary to ensure that DNA is extracted in appropriate amounts and that extracted DNA is inhibitor-free. This study aimed at selecting a single protocol suitable for the extraction of total DNA from Gram positive and negative bacteria isolated from different sources, as well as a protocol for the direct extraction of DNA from soil. Four experimental protocols and a commercial kit were tested for the extraction of total DNA from isolated bacteria. Among the protocols, the detergent + salt + thermal incubation method (based on Harju et al., 2004) was considered the most promising because it produced satisfactory yields of DNA, with adequate quality for all isolates studied, especially Staphylococcus aureus, without the need to use enzymes and glass beads which can make the extraction process more expensive. Three experimental protocols and the commercial kit were tested for the direct extraction of DNA from soil. Regarding PCR amplification, the amount of total DNA extracted is less limiting than its quality. Thus, commercial kit PowerMax™ Soil DNA Isolation (MoBio) offered more promising results, because although this provided low yields of DNA, it was sufficient for polymerase chain reaction (PCR) amplification.