The combination of daptomycin and fosfomycin is attracting attention due to the rising resistance observed in methicillin-resistant (MRSA). Most studies indicate that this combination exhibits synergistic or additive antimicrobial activity against MRSA. However, its capacity to prevent resistance remains uncertain. In this study, we first investigated the antibacterial effect of daptomycin and fosfomycin on MRSA, followed by predicting MRSA resistance under the influence of different drugs using a resistance mutation selection window. Subsequently, we conducted preliminary analyses of drug resistance gene mutations in resistance mutants through gene sequencing technology. Additionally, we established an biofilm infection model to explore the internal tolerance associated with phenotypic alterations in biofilms and assess the combination's ability to prevent drug resistance. Combination drug sensitivity experiments demonstrated that daptomycin and fosfomycin synergistically enhanced antimicrobial effects against the tested strains. This combination reduced the mutant prevention concentration of each drug and narrowed the selection window for drug-resistant mutations. Sequencing results indicated that specific resistance genes were mutated in the single-drug mutants, while no mutations were detected in the combination. Furthermore, the combination exhibited stronger inhibition and removal of biofilm compared to single agents. In conclusion, the daptomycin-fosfomycin combination displays a synergistic antibacterial effect against MRSA and shows enhanced capacity to prevent bacterial resistance, likely attributed to its ability to inhibit resistance gene mutations and exhibit superior anti-biofilm activity.