Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic strains. as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of as in vivo model for pre-clinical studies of natural substances.