Background: Early gut microbiome development is critical for neonatal health, and its dysbiosis may impact long-term animal productivity. This study examined the effects of parenteral Ceftiofur Crystalline Free Acid (CCFA) on the composition and diversity of the neonatal lamb fecal microbiome. Additionally, the emergence of antimicrobial resistance genes associated with CCFA exposure was investigated. Methods: Sixteen healthy neonatal lambs were randomly assigned to CCFA-treated (n = 8) or control (n = 8) groups. Fecal samples were collected on days 0, 7, 14, 28, and 56. Genomic DNA was extracted and sequenced using the Illumina MiSeq platform. Microbial composition was analyzed using the MG-RAST pipeline with the RefSeq database. Results: There were distinct microbial populations in the CCFA-treated lambs compared to the control group at each time point, with a highly significant decrease in alpha and beta diversity. The CCFA treatment showed a reduction in several key microbial taxa during nursing, but these differences were diminished by day 56. Unlike the control group, CCFA-treated lambs had core microbes potentially carrying multiple antibiotic resistance genes, including those for beta-lactam, fosfomycin, methicillin, and multidrug resistance. Conclusion: The early sheep fecal microbiome demonstrated resilience, repopulating after CCFA-induced perturbation despite a temporary reduction in key taxa during the nursing period. This highlights the microbiota's stability following a short-course antibiotic challenge. However, the transient disruption suggests potential negative impacts of antibiotics on the early gut microbiome. Notably, CCFA resistance persisted, raising concerns about its possible spread to the surrounding environment.