Spinal cord injury (SCI) is a devastating neurological state that could lead to motor, sensory, and autonomic dysfunction. In addition to its direct impact on the central nervous system, SCI exerts systemic effects, including disruption of gut homeostasis and alterations in the gut microbiota, which can contribute to sustained inflammation and hinder functional recovery. While antibiotic administration during the acute phase of SCI is clinically indicated, it may exacerbate microbial dysbiosis. In this study, we investigate the combined effects of SCI and cefazolin treatment on the gut microbiome of female rats. Animals were assigned to three groups: NAÏVE (no intervention), SHAM (cefazolin only), and INJURY (T10 spinal cord contusion plus cefazolin). Cefazolin was administered for seven days after the injury, fecal samples were collected at baseline (day 0), and on days 7, 14, 21, and 28 post-SCI. DNA was extracted and subjected to 16S rRNA gene amplicon sequencing, followed by bioinformatic analysis. Our findings revealed significant microbial dysbiosis in the INJURY group, including reduced alpha diversity and distinct shifts in microbial composition. These changes were most prominent during the acute phase post-SCI. Our findings highlight the compounding effects of spinal trauma and antibiotic exposure on the gut microbiome and underscore the importance of microbial stability in SCI recovery.