Background <p>Certification and disease management practices are reliant on accurate pathogen detection assays. Effective nucleic acid extraction is the cornerstone of various molecular detection techniques, and many virus and viroid RNA extraction protocols have been developed, each with accompanying advantages and limitations. The extraction method influences the type, quantity and quality of RNA and, therefore, the overall sensitivity of the assay.</p> Methods <p>Plants from two citrus types were infected with multiple viruses and viroids. Ten plants from each were selected. RNA was extracted from bark and leaf samples using four protocols: CL (CTAB/LiCl), CE (CTAB/ethanol), TL (acid phenol buffer/LiCl and isopropanol) and SI (SDS/isopropanol). Extracts were subjected to RT-qPCR for pathogen quantitation. Results were confirmed with Illumina high-throughput sequencing (HTS). Leaf material from a citrus type was resampled, RNA extracted with CE, SI and CP (CTAB/isopropanol) and pathogen quantities determined with RT-qPCR. An additional five plants were also selected and subjected to RNA extraction with CP, SI and a modified CE. RT-qPCRs and HTS were applied to all of these extracts. Protocols were evaluated based on pathogen quantities and various bioinformatic approaches. Reference genes and CTV genotypes were also assessed with HTS.</p> Results <p>The extraction protocol had a remarkable influence on RT-qPCR and HTS pathogen detection ability. The CL method performed best for CTV while SI proved more sensitive for the other viruses. Viroid detection had increased variability with CE extracts containing more viroid RNA although CL extracts showed comparable amounts of CDVd. The CE protocol indicated potential debilitating effects on virus detection. The additional sampled plants subjected to the modified CE protocol, showed increased virus and decreased viroid amounts. The extraction protocol did, however, not significantly influence CTV genotype composition and the investigation of reference genes provided insight into appropriate genes to be used as internal controls for cross sample comparisons.</p> Conclusions <p>Despite the differential pathogen extraction amongst protocols, all target pathogens could be detected with appropriate RT-qPCR replicates and combined bioinformatic approaches. This study emphasises that although multiple protocols can be effective, further selection and optimisation could enhance the performance. Present research serves as a foundation for the refinement of diagnostic pipelines in citrus pathology.</p>