Introduction: The purity of genomic DNA (gDNA) extracted from different clinical specimens optimizes sensitivity of polymerase chain reaction (PCR) assays. This study attempted to compare two different DNA extraction techniques namely salting-out and classic phenol-chloroform. Materials and Methods: Qualification of two different DNA extraction techniques for 634 clinical specimens highly suspected of having mycobacterial infection was performed. Genomic DNA was extracted from 330 clinical samples using phenol-chloroform and 304 by non-toxic salting-out. Qualification of obtained gDNA was done through amplification of internal controls, β-actin and β-globin. Results: β-actin-positive was detected in 279/330 (84%) and 272/304 (89%) samples by phenol-chloroform technique and salting-out, respectively. PCR inhibitor was found for the gDNA of 13/304 (4%) patient samples were negative by β-actin and β-globin tests via salting-out technique in comparison with gDNAs from 27/330 (8.5%) samples extracted by phenol-chloroform procedure. No statistically significant difference was found between phenolchloroform technique and salting-out for 385 sputum, 29 bronchoalveolar lavage (BAL), 105 gastric washing, and 38 body fluid (P=0.04) samples. This illustrates that both techniques have the same quality for extracting gDNA. Conclusion: This study discloses salting-out as a non-toxic DNA extraction procedure with a superior time-efficiency and cost-effectiveness in comparison with phenol-chloroform and it can be routinely used in resource-limited laboratory settings.