The rapid evolution of forensic molecular biology has shown the utility of the oral microbiota as a consistent biomarker for body fluid identification. This study assessed the use of quantitative real-time polymerase chain reaction (qPCR) to detect three oral bacterial species, Streptococcus salivarius, Streptococcus sanguinis, and Neisseria subflava, for forensic saliva identification and sex determination. A cross-sectional descriptive study was performed in 45 healthy male and female volunteers aged 18–24 years at the School of Medicine, Badr University, in Cairo. DNA was isolated from saliva, nasal fluid, and blood samples and assessed via qPCR. The expression levels of the three bacterial strains were much greater in saliva than in nasal fluid or blood (p < 0.001), validating their specificity for the oral microbiome. A discriminant function based on S. sanguinis and N. subflava correctly classified sex with an overall accuracy of 75.6%, with S. sanguinis showing statistically significant contribution and N. subflava showing borderline significance (p = 0.05). The results confirmed that qPCR of specific oral bacterial DNA provides a sensitive and specific test for the identification of saliva with good prospects for sex determination in forensic science. Further large-scale validation studies are suggested to ensure its applicability in actual forensic environments.