Musculoskeletal infections (MSKIs) pose significant challenges in orthopedic surgery. These infections are often caused by S. epidermidis and S. epidermidis. Early diagnosis is crucial, as it allows prompt treatment before the infection becomes chronic and more difficult to manage. This study evaluated the expression of microRNAs (miRNAs) in the serum of patients with staphylococcal MSKI and their potential as biomarkers. Serum samples from patients with confirmed staphylococcal MSKI were used for RNA sequencing and validation (n = 24 S. aureus, n = 25 S. epidermidis and n = 25 controls). In S. aureus-infected patients compared to uninfected controls, 23 miRNAs were upregulated and 20 downregulated, while 94 miRNAs were upregulated and 31 downregulated in those infected with S. epidermidis. Validation experiments revealed the upregulation of hsa-miR-1246, hsa-miR-483-5p, and hsa-miR-1290, and the downregulation of hsa-miR-1-3p, hsa-miR-148b-3p, and hsa-miR-23a-3p in S. aureus-infected patients compared to both S. epidermidis-infected patients and uninfected controls. Human peripheral blood mononuclear cells treated in vitro with bacterial culture supernatants exhibited a similar pattern of miRNA expression. Receiver operating characteristic (ROC) curve analyses demonstrated that combining four miRNAs (miR-1246, miR-1290, miR-148b-3p, and miR-23a-3p) achieved a high diagnostic accuracy, with an area under the curve of 0.96, distinguishing S. aureus from S. epidermidis-infected patients. In summary, this study identified four differentially expressed miRNAs in S. aureus-infected patients compared to those with S. epidermidis infections and uninfected controls. These findings allow miRNA to be used as MSKI markers and to study their effect on immune responses during bacterial infections.