Identifying promising probiotic candidates for further development remains challenging. Traditional mammalian models are invaluable for assessing efficacy, but the associated limitations slow the preliminary screening process. The larvae have emerged as a powerful screening model. Most probiotic studies on use intra-hemocelic injection, bypassing the natural gut entry point, making it analogous to introducing probiotics into the mammalian blood. Therefore, despite their advantages, discrepancies exist between and mammalian models, particularly regarding the route of probiotic administration. This study bridges this gap by investigating the differential effects of the commonly studied intra-hemocelic injection and the less common oral administration of a probiotic, Nissle 1917 (EcN1917), on its protective efficacy against a gastrointestinal pathogen, Typhimurium ATCC 14028 (ST14028). This study demonstrated that oral EcN1917 pre-treatment significantly increased survivability against ST14028 infection compared to the control group and alleviated the pathogen gut burden. Notably, injection pre-treatment decreased survivability. This discrepancy is attributed to the dual nature of probiotics, exhibiting beneficial effects in the gut but acting as pathogens in non-native locations like the hemolymph, concluding that the route of probiotic administration in significantly impacts the protective effects of probiotics.