BackgroundAntimicrobial alternatives are urgently needed, including for poultry production systems. In this study, we tested the potential broad-range antimicrobial alternative peracetic acid, delivered in feed via the hydrolysis of encapsulated precursors through a 28-day study using 375 Ross 308 broiler chickens. We tested two peracetic acid concentrations, 30 and 80 mg/kg on birds housed on re-used litter, and we evaluated the impact of both levels on gut microbial communities, bacterial concentration, antimicrobial resistance genes relative abundance and growth performance when compared to control birds housed on either clean or re-used litter.ResultsBody weight gain and feed conversion ratio improved in peracetic acid fed birds. At d 28, birds given 30 mg/kg of peracetic acid had a decreased Firmicutes and an increased Proteobacteria abundance in the jejunum, accompanied by an increase inBacillus,FlavonifractorandRombustiain the caeca, and a decreased abundance of tetracycline resistance genes. Chicken given 80 mg/kg of peracetic acid had greater caecal abundance of macrolides lincosamides and streptogramins resistance genes. Growth performance on clean litter was reduced compared to re-used litter, which concurred with increased caecal abundance ofBlautia, decreased caecal abundance ofEscherichia/Shigella, AnaerostipesandJeotgalicoccus, and greater gene abundance of vancomycin, tetracycline, and macrolides resistance genes.ConclusionsPeracetic acid could be used as a safe broad-spectrum antimicrobial alternative in broilers. Encapsulated precursors were able to reduce the bacterial concentration in the jejunum whilst promoting the proliferation of probiotic genera in the caeca, especially at the low peracetic acid concentrations tested, and improve growth performance. Moreover, our findings offer further insights on potential benefits of rearing birds on re-used litter, suggesting that the latter could be associated with better performance and reduced antimicrobial resistance risk compared to clean litter rearing.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-023-00881-w.