New biocidal solutions are needed to combat effectively the evolution of microbes developing antibiotic resistance while having a low or no environmental toxicity impact. This work aims to assess the efficacy of commonly used biocides and natural-based compounds on the disinfection of silicone and stainless steel (SS) surfaces seeded with different Staphylococcus aureus strains. Minimum inhibitory concentration was determined for synthetic (benzalkonium chloride-BAC, glutaraldehyde-GTA, ortho-phthalaldehyde-OPA and peracetic acid-PAA) and natural-based (cuminaldehyde-CUM), eugenol-EUG and indole-3-carbinol-I3C) biocides by the microdilution method. The efficacy of selected biocides at MIC, 10 × MIC and 5500 mg/L (representative in-use concentration) on the disinfection of sessile S. aureus on silicone and SS was assessed by viable counting. Silicone surfaces were harder to disinfect than SS. GTA, OPA and PAA yielded complete CFU reduction of sessile cells for all test concentrations as well as BAC at 10 × MIC and 5500 mg/L. CUM was the least efficient compound. EUG was efficient for SS disinfection, regardless of strains and concentrations tested. I3C at 10 × MIC and 5500 mg/L was able to cause total CFU reduction of silicone and SS deposited bacteria. Although not so efficient as synthetic compounds, the natural-based biocides are promising to be used in disinfectant formulations, particularly I3C and EUG.