The global rise of antibiotic resistance necessitates novel therapeutic strategies for infectious diseases. Inhibition of bacterial efflux pumps, which contribute to multidrug resistance, represents a promising approach to restore or even increase the efficacy of existing antibiotics. Using fluorescence-based ethidium bromide accumulation, broth microdilution, and checkerboard assays, we evaluated 26 endogenous steroidal hormones and neurosteroids, along with 30 synthetic derivatives, for their ability to enhance antibiotic susceptibility in multidrug-resistant Staphylococcus aureus. Structure-activity relationship analysis identified compounds 13 and 16 as lead candidates, exhibiting strong efflux pump inhibition and marked reductions in the minimum inhibitory concentrations of ciprofloxacin and erythromycin. Both compounds showed additive effects in checkerboard assays. Modifications at C-3 (polar substitution) and C-17 (3α,5β-stereochemistry and nonpolar substitution) were essential for potent efflux inhibition and sensitization, although these modifications were not additive when combined. Transcriptome analysis further revealed that compound 13 significantly downregulated S. aureus virulence-associated genes when administered alone or in combination with antibiotics. Cytotoxicity assessment in human peripheral blood mononuclear cells and receptor transactivation assays for estrogen, androgen, and progesterone receptors indicated that the most active derivatives were non-toxic and lacked detectable endocrine activity, suggesting a favorable safety profile. Overall, these findings support the concept that rationally designed andostane-based steroidal scaffolds can function as competitive bacterial efflux pump inhibitors and serve as potential antibiotic adjuvants to mitigate efflux-mediated resistance.