Watermelon seedling blight and fruit blotch caused by Acidovorax citrulli (Ac) is a seed-borne bacterial disease. The bacterium survives in/on seeds for years. The aim of this study was to determine the most favourable, reliable and fast method for detection Ac from cucurbit seeds. Cucurbit seeds were homogenised using a blender and left in orbital shaker for 24 h in a nutrient broth. The methods seedling grow out, pathogen isolation from seed washing water dilutions onto semi selective media P-278 and mEBB and general medium King B, DAS-ELISA and BIO-PCR were used for detection. Water-soaked lesions observed on cotyledons. Since DAS-ELISA and PCR tests with seed dilutions onto King B has failed to detect Ac from seed lots, Ac specific PCR with dilutions from semi selective media has successfully detect the pathogen. As a result of this study, four (one cucumber, one melon and two watermelon seed lots) out of twenty two tested cucurbit seeds were contaminated with Ac. This study recommended that this BIO-PCR is safe, reliable and fast for detection Ac from cucurbit seeds without any seedling growth.