Antibiotics misuse in humans and animals accelerated global emergence and dissemination of antimicrobial resistance, that nowadays is a threat to global health. Bacteria harboring antibiotic resistance genes (ARG) are known to be resident in the intestinal microbiota (IM) of mammals since birth, constituting the resistome. Calves represent a special concern for the high prevalence of ARGs within the IM. Antibiotics are major disruptors of the IM, depleting bacterial species benefic for the host health mostly and favoring the emergence of potential pathogens. The antibiotic pressure plays a major role on the selection of resistant bacteria within the gut, that then could potentially contaminate the environment, increasing the potential dissemination to other hosts (humans or animals) through fecal-oral route or the food chain. Amoxicillin is one of the most used antibiotics worldwide in human and veterinary medicine. The effect of this antibiotic was investigated in the IM from multiple hosts and highlighted an increase of determinants conferring resistance to amoxicillin and effects on the IM composition. Despite the predominant usage and the important role of amoxicillin in veterinary medicine, longitudinal studies analyzing the effect of amoxicillin treatment on the intestinal microbiota of calves are lacking. To fill in this gap of knowledge, the effect of amoxicillin treatment was analyzed in a group of calves hosted in commercial farms and suffering from omphalitis via three axes. First, qPCR assays targeting the major ARGs found in Enterobacterales and also integrons (intI1/2/3) were designed and validated. These assays were used to quantify ARGs in stools of calves collected before amoxicillin treatment, at the end of the amoxicillin therapy, and one week after the end of the therapy. The amount of the blaTEM gene, responsible for amoxicillin resistance, increased after amoxicillin treatment and decreased after therapy withdrawal in treated calves. Other genes followed the same dynamic as blaTEM suggesting a potential co-selection by amoxicillin of resistance to other classes of antibiotics. Amoxicillin treatment selected amoxicillin-resistant Gram-negative bacteria mainly represented by E. coli, according to cultivation and enumeration on plates amended with amoxicillin or without the antibiotic. Amoxicillin-resistant, Gram-negative bacteria increased after amoxicillin therapy in treated calves and decreased one week after withdrawal. One-hundred-fifty-two isolates were analyzed for their susceptibility and their genome was sequenced. Globally, phylogroup A was the most represented in the collection (n = 57/152), followed by phylogroup B1, D and B2. The most frequent STs associated to the isolates belonged to CC10 (ST10/34/48/167), followed by CC23 (ST23 and ST88), and CC155 (ST56 and ST58). The blaTEM-1A/B gene was present in all amoxicillin-resistant E. coli (n = 114/152). This gene was often associated to IS26 and IS91 and located close to a mer operon. blaTEM-1was also found close to aminoglycosides and tetracycline resistance genes. Class 1 integrons was found in most of the isolates and ten variants were detected. The most represented plasmids in the isolates were IncFII, IncFIA-IB, and IncQ1 type. Preliminary results from the analysis of DNA extracted from stools the by shotgun sequencing, suggested a decrease, at the end of the treatment, of the Firmicutes and Verrucomicrobia phyla, whereas Bacteroidetes, Actinobacteria and Proteobacteria abundance increased. Integrating multiple methodological axes, our study pave the way to understand the role of amoxicillin therapy in the IM composition and resistances of calves. These combined approaches are necessary to understand the global effect of antibiotic therapy for improving antibiotic usage and find strategies to limit the negative consequences of antibiotic therapies on the host health and to counteract the selection of ARGs in the gut.