Signaling between intestinal microbiota and the brain influences neurologic outcome in multiple forms of brain injury. The impact of gut microbiota following traumatic brain injury (TBI) has not been well established. Our objective was to compare TBI outcomes in specific pathogen-free mice with or without depletion of intestinal bacteria. Adult male C57BL6/J SPF mice (n = 6/group) were randomized to standard drinking water or ampicillin (1 g/L), metronidazole (1 g/L), neomycin (1 g/L), and vancomycin (0.5 g/L) (AMNV) containing drinking water 14 days prior to controlled cortical impact (CCI) model of TBI. 16S rRNA gene sequencing of fecal pellets was performed and alpha and beta diversity determined. Hippocampal neuronal density and microglial activation was assessed 72 h post-injury by immunohistochemistry. In addition, mice (n = 8–12/group) were randomized to AMNV or no treatment initiated immediately after CCI and memory acquisition (fear conditioning) and lesion volume assessed. Mice receiving AMNV had significantly reduced alpha diversity (p < 0.05) and altered microbiota community composition compared to untreated mice (PERMANOVA: p < 0.01). Mice receiving AMNV prior to TBI had increased CA1 hippocampal neuronal density (15.2 ± 1.4 vs. 8.8 ± 2.1 cells/0.1 mm; p < 0.05) and a 26.6 ± 6.6% reduction in Iba-1 positive cells (p < 0.05) at 72 h. Mice randomized to AMNV immediately after CCI had attenuated associative learning deficit on fear conditioning test (%freeze Cue: 63.7 ± 2.7% vs. 41.0 ± 5.1%, p < 0.05) and decreased lesion volume (27.2 ± 0.8 vs. 24.6 ± 0.7 mm³, p < 0.05). In conclusion, depletion of intestinal microbiota was consistent with a neuroprotective effect whether initiated before or after injury in a murine model of TBI. Further investigations of the role of gut microbiota in TBI are warranted.