Background: Perturbations of animal-associated microbiomes from chemical stress can affect host physiology and health. While dysbiosis induced by antibiotic treatments and disease are well known, chemical, non-antibiotic drugs have recently been shown to induce changes in microbiome composition, warranting further exploration. Loperamide is an opioid-receptor agonist drug widely prescribed drug for treating acute diarrhea in humans. Loperamide is also used as a tool to study the impact of bowel dysfunction in animal models by inducing constipation, but its effect on host-associated microbiota is poorly characterized. Results We used conventional and gnotobiotic larval zebrafish models to show that in addition to host-specific effects, loperamide also has anti-bacterial activities that directly induce changes in microbiota diversity. This dysbiosis is due to changes in bacterial colonization, since germ-free zebrafish mono-reconventionalized with bacterial strains sensitive to loperamide are colonized up to 100-fold lower when treated with loperamide. Consistently, the bacterial diversity of gnotobiotic zebrafish colonized by a mix of representative bacterial strains is affected by loperamide treatment. Conclusion Our results demonstrate that loperamide, in addition to host effects, also induces dysbiosis in a vertebrate model, highlighting that established treatments can have underlooked secondary effects on microbiota structure and function. This study further provides a insights for future studies exploring how common medications directly induce changes in host-associated microbiota. Graphical Abstract