The combination of levulinic acid (LVA) and sodium dodecyl sulfate (SDS) in recent years has shown a considerable potential to use as an antimicrobial intervention. The objectives of this study were to evaluate the antimicrobial efficacy of the combination against Staphylococcus aureus in both planktonic and biofilm states and to investigate the transcriptional changes in S. aureus biofilms coincubated with sublethal concentrations of LVA and/or SDS. The minimum inhibitory concentrations (MICs) of LVA and SDS determined by the microdilution method were 3.125 and 0.039 mg/mL, respectively. An additive bacteriostatic interaction (fractional inhibitory concentration index = 1) between the two compounds was observed by the checkerboard assay, whereas a synergistic bactericidal activity was displayed by the time-kill assay. The biomass and viable cells in the biofilms were reduced by both antimicrobials either alone or in combination in a dose-dependent manner. Transcriptomics indicated that more differentially expressed (DE) genes were observed in the biofilm treated with SDS (103 up- and 205 downregulated DE genes) and LVA + SDS (187 up and 162 down) than that coincubated with LVA (34 up and 32 down). The SDS and LVA + SDS treatments mainly affected the expression of genes responsible for cell surface proteins, virulence factors, adhesins, and capsular polysaccharides. Both the antibiofilm assay and the transcriptomics indicated that SDS, not LVA, was the major chemical contributing to the antibacterial efficacy of the combination. This study reveals the behavioral responses and protective mechanisms of S. aureus to LVA and SDS applied individually or in combination.