Background: Neonatal Calf Diarrhea (NCD) is a critical disease contributing to neonatal mortality in calves, with its pathogenesis remaining to be fully understood.</p><p>Results: Our study was conducted in Inner Mongolia, a key region for cattle farming in China, aiming to provide a comprehensive overview of the epidemiology and treatment of calf diarrhea in this locale. No significant correlation was found between the incidence of diarrhea and sampling points or medication. Diarrhea cases were stratified into five severity levels based on fecal characteristics, with L1 representing the least severe, closest to normal stool consistency, and L5 indicating the most severe diarrhea. To elucidate the pathogenesis of NCD, we performed 16S rRNA and metagenomic sequencing analyses across the four categorized levels. Microbial diversity analyses revealed distinct variations in the microbial communities among the different severity levels. Employing binning and LEfSe methodologies, we identified a bacterial pathogen from the family f_Streptococcaceae and a viral agent, Escherichia phage VpaE1_ev108, both of which showed significant associations with disease progression. Gene function enrichment analysis uncovered a broad spectrum of antibiotic resistance genes, even in farms without direct antibiotic treatment, underscoring the pervasive presence of drug resistance. Phage therapy, with its potential to reduce biological contamination and resistance on farms, holds significant promise for future applications.</p><p>Conclusions: this study conducted a regional analysis of incidence and treatment of NCD across 12 farms in the Inner Mongolia Autonomous Region, China. Concurrently, potential pathogenic bacteria associated with NCD were identified via 16S rRNA and metagenomic sequencing analyses: Streptococcus. Furthermore, we assembled multiple relatively complete binned bacterial and viral genomic datasets. These datasets provide a rich resource for researchers, offering valuable insights into the complex etiology of NCD.