The success of Acidovorax citrulli detection by seed health testing of commercial cucurbitaceous seeds depends on the efficiency of pathogen extraction. In the present study, different extraction techniques were compared to identify factors that influence A. citrulli detection in naturally contaminated cucurbit seeds. Embryo-infected watermelon seeds, produced by pistil inoculation, were mixed with non-infected seeds to generate samples (n = 1000 seeds) with low infection levels (approximately 104 cells per sample). Additionally, two naturally infested melon seed lots were tested. A. citrulli was extracted from seeds by soaking or crushing, followed by one or two centrifugation steps. Samples extracted by soaking seeds yielded better amplification efficiency (103%) compared with crushing (93%), as determined by quantitative real-time PCR analysis. This was most likely due to a reduction of the concentration of inhibitors present in the DNA samples. PCR assays using three different A. citrulli-specific primer sets highlighted that soaking followed by two centrifugation steps enhanced pathogen detection (100% of the samples) and the mean cycle threshold (Ct) value was significantly lower than those observed for the other pathogen extraction techniques. These results indicate that the optimised extraction protocol combined with PCR analysis can improve routine seed health testing for A. citrulli.