Simple SummaryBovine respiratory disease (BRD) affects the lower respiratory tract of cattle, causing high mortality. The syndrome has a multifactorial etiology and transport seems to favor pathogen proliferation. This study investigated the prevalence of different pathogens involved in BRD, in the nasal microbiota of beef steers collected before and after a long-distance journey. A total of 56 Limousine animals were included, travelling in three different shipments, on the same route from France to southern Italy in a semitrailer, on three different days from February to April. Prior to shipment (T0) and four days after arrival (T1), two deep nasopharyngeal swabs (DNS)/steer were collected and tested by bimolecular analysis. Neither bovine viral diarrhea virus (BVDV) nor bovine herpesvirus type 1 (BoHV-1) were detected. A higher prevalence of Histophilus somni was observed in the DNS collected during the third shipment in comparison with those registered during the first and the second one, probably due to a higher prevalence at departure. Conversely, the prevalence of bovine coronavirus, bovine respiratory syncytial virus, Mannheimia haemolytica, Mycoplasma bovis and Pasteurella multocida was higher on arrival in comparison with departure, confirming data reported in the literature. Overall, there were nasal microbiota changes in beef steers, with an increase in the prevalence of pathogens associated with BRD after travelling.Bovine respiratory disease (BRD) is a serious health and economic problem in the beef industry, which is often associated with transportation and caused by different pathogens. The prevalence of bovine herpesvirus type 1 (BoHV-1), bovine adenovirus (BAdV), bovine viral diarrhea virus (BVDV), bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus (BPiV), Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, in the nasal microbiota of beef steers before and after the same long-distance journey from France to southern Italy was documented. Fifty-six Limousine animals of three different shipments, travelling on three different days from February to April, were included. Prior to shipment (T0) and four days after arrival (T1), two DNS/animal were collected and tested by Real Time quantitative PCR (qPCR). Univariate logistic regression was carried out, considering time and day as fixed factors and the outcome of qPCR for each pathogen as a dependent categorical dichotomous variable (positive/negative, 1/0). The fact that the number of H. somni positive animals were found to be higher in the third shipment than the first and second one, indicating that this pathogen was already present before loading, is relevant. The prevalence of BCoV, BRSV, M. haemolytica, M. bovis, P. multocida was higher at T1 than T0, suggesting that other factors, such as stress and the epidemiological status of the arrival farm, played a role. The tested animals were not treated before and after transport, and our results are in agreement with the current literature, supporting the hypothesis that the prevalence of pathogens related to BRD would increase after travelling, with an increased risk of pathogens shedding.