Australia depends heavily on the importation of vegetable seeds for crop production, with large quantities entering the country annually. However, the global movement of seeds has amplified the spread of seed-borne pathogens, including fungi, bacteria, viruses, viroids, and oomycetes, significantly increasing biosecurity risks. The international development, multiplication, and processing of seeds across multiple countries further heightens the potential for exposure to novel pathogens. In response, the Australian Department of Agriculture, Fisheries and Forestry (DAFF) has implemented emergency measures requiring pre-border or border testing for specified pathogens in selected vegetable seed species, successfully reducing viroid contamination in imported solanaceous seed lots from 10% in 2013 to less than 1.5% in 2021. To address the continued risk of exotic pathogen introduction, DAFF has initiated a review of import conditions for four key vegetable seed families—Apiaceae, Brassicaceae, Cucurbitaceae, and Solanaceae—that are critical to Australia’s food security and export economy. This project seeks to develop a cost-effective, adaptable, high-throughput molecular diagnostic system for detecting exotic and regulated pathogens in seeds. The system will incorporate novel targeted approaches to provide equivalence to current gold-standard testing methods while improving speed and efficiency. Currently, seed testing protocols require large sample volumes and separate molecular methods for DNA and RNA pathogens, which increases both time and costs. To streamline these processes, the project aims to develop a universal nucleic acid extraction protocol capable of efficiently isolating quality DNA and RNA for subsequent high-throughput screening (HTS) method development. For this study, cucumber mottle mosaic virus (CGMMV) and melon necrotic spot virus (MNSV), which are high-priority pathogens for nine cucurbitaceous vegetable species, were selected for evaluation. Initially, 56 potential nucleic acid extraction methods were identified and narrowed down to 23 based on buffer composition and effectiveness. Nucleic acids were extracted from cucurbit seeds spiked with known quantities of synthetic RNA and DNA from CGMMV, MNSV, and Acidovorax citruli (AC), revealing that SDS-based buffers specifically S2 (Oñate-Sánchez et al., 2008) and S4 (Mayjonade et al., 2018), provided the highest DNA and RNA recovery rates, outperforming current ELISA extraction buffers. Further evaluations with additional seed types are ongoing to finalise a universal and cost-effective method for nucleic acid extraction and pathogen detection. This extraction method will serve as a unified source of DNA and RNA, facilitating the development of novel pathogen detection technologies while reducing the time and cost of current extraction protocols, ultimately improving biosecurity and protecting Australian agriculture from emerging biosecurity threats.