Simple SummaryThere has been extensive research addressing the positive effects of medicinal plant extracts using food fish as animal models. The present research was an attempt to assess zebrafish viability, considering the 3Rs approach, as an animal model for dietary experiments for fish nutrition studies, particularly evaluating novel additives for further implications in food fish. We have found oregano (Origanum vulgare) extract to be remarkably effective on fish growth and the fish immune system, enabling fish to fight a bacterial invasion. This study evaluated the dietary effects of an ultrasound-assisted extract of Origanum vulgare on the growth, antioxidant and immune responses (serum and mucosal) and resistance of zebrafish (Danio rerio) against Aeromonas hydrophila. Four hundred and forty adult zebrafish were distributed into 12 tanks and fed 4 experimental diets including 0% (control), 0.5% (M1), 1% (M2) and 2% (M3) of the extract per kg−1 diet for eight weeks. Fish were then challenged with A. hydrophila and mortality was recorded for 10 days. Results revealed that the extract exerted potent effects on growth parameters of weight gain and specific growth rate. The feed conversion ratio was significantly lower in fish fed extract-incorporated diets. O. vulgare extract improved antioxidant and immune responses, resulting in less sensitivity to oxidative stress and a higher survival rate when challenged with A. hydrophila. Overall, the greatest effects were observed in individuals with 1% dietary inclusion of the extract. These results suggest that the extract from the plant Origanum vulgare possesses a great potential to be used in the aquaculture industry and that zebrafish is an appropriate model for nutrition studies.