The evolution of antimicrobial-resistant pathogens is a global health and development threat. Nanomedicine is rapidly becoming the main driving force behind ongoing changes in antimicrobial studies. Among nanoparticles, silver (AgNPs) have attracted attention due to their versatile properties. The study aimed to investigate the effects of AgNPs and L-carnitine (LC) on mixed Candida albicans and Staphylococcus aureus in the mice vaginitis model. Study of antimicrobial activity of AgNPs evaluated by Minimum Inhibitory Concentration (MIC) and Minimum Biocidal Concentration (MBC) assays. AgNPs inhibited biofilm formation of microbial strains, which was tested by using crystal violet staining. In the current study, we evaluated the effects of AgNPs and LC in NMRI mice infected intravaginally with C. albicans/ S. aureus for two weeks. The proportion of mice in each stage of the estrous cycle (proestrus, estrus, metestrus, and diestrus) was examined. Histological properties were assessed by hematoxylin/ eosin (H&E) staining of formalin-fixed, paraffin-embedded vaginal tissue sections. Based on the results, MICs of AgNPs against S. aureus, C. albicans, and their combination were 252.3, 124.8, and 501.8ppm, and their minimum biofilm inhibitory concentration (MBIC) was 500, 250, and 1000ppm, respectively. The estrous cycle in the treated group was similar to the control. Vaginal histology and cytology showed that LC can improve tissue damages caused by vaginitis and AgNPs. This study demonstrates the promising use of AgNPs as antimicrobial agents and the combination of AgNPs/ LC could be a great future alternative in the control of vaginitis.