The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.