The discovery of the DNA structure sparked significant advances in molecular biology, including the Polymerase Chain Reaction (PCR) technique. PCR allows for the amplification of specific DNA sequences, which is essential for molecular research and diagnostics. Primer design is crucial to ensure the specificity and sensitivity of assays being performed in silico and experimentally validated.This study aims to present the OligoDesign tool (license number 512022000569-7 by the Brazilian National Institute of Industrial Property [INPI]), which seeks to optimize primer design for PCR, with an initial focus on validating primers for Staphylococcus aureus and future primers targeting oncogenes. The software was developed in Java and allows the selection of target and non-target sequences to identify regions with genetic differences, using the Myers algorithm. In silico validation was performed to check for secondary structure formation and primer specificity, while experimental validation was carried out by qPCR with DNA samples extracted from bacterial cultures and contaminated food. The results demonstrated good amplification efficiency and high specificity for S. aureus, with 76.2% exclusivity compared to other microorganisms. The OligoDesign tool demonstrated high efficiency in primer design for S. aureus. Building on this success, it will now be used for the C-KIT oncogene, with the aim of updating and refining the system to make it available to other users in a web-based format.