Periodontitis (PD) is a polymicrobial dysbiotic immuno-inflammatory disease. It is more prevalent in males and has poorly understood pathogenic molecular mechanisms. Our primary objective was to characterize alterations in sex-specific microRNA (miRNA, miR) after periodontal bacterial infection. Using partial human mouth microbes (PAHMM) (Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in an ecological time-sequential polybacterial periodontal infection (ETSPPI) mouse model, we evaluated differential mandibular miRNA profiles by using high-throughput Nanostring nCounter® miRNA expression panels. All PAHMM mice showed bacterial colonization (100%) in the gingival surface, an increase in alveolar bone resorption (p < 0.0001), and the induction of a specific immunoglobin G antibody immune response (p < 0.001). Sex-specific differences in distal organ bacterial dissemination were observed in the heart (82% male vs. 28% female) and lungs (2% male vs. 68% female). Moreover, sex-specific differential expression (DE) of miRNA was identified in PAHMM mice. Out of 378 differentially expressed miRNAs, we identified seven miRNAs (miR-9, miR-148a, miR-669a, miR-199a-3p, miR-1274a, miR-377, and miR-690) in both sexes that may be implicated in the pathogenesis of periodontitis. A strong relationship was found between male-specific miR-377 upregulation and bacterial dissemination to the heart. This study demonstrates sex-specific differences in bacterial dissemination and in miRNA differential expression. A novel PAHMM mouse and ETSPPI model that replicates human pathobiology can be used to identify miRNA biomarkers in periodontitis.