The health benefits of garlic and other organosulfur-containing foods are well recognized and have been attributed to both pro-oxidant and antioxidant activities. The effects of garlic are surprisingly similar to those of hydrogen sulfide (H₂S) which is also known to be released from garlic under certain conditions. However, recent evidence suggests that polysulfides, not H₂S, may be the actual mediator of physiological signaling. In this study we monitored formation of H₂S and polysulfides from garlic oil in buffer and in HEK293 cells with fluorescent dyes, AzMC and SSP4, respectively and redox activity with two redox indicators roGFP and DCF. Our results show that H₂S release from garlic oil in buffer requires other low molecular weight thiols such as cysteine (Cys) or glutathione (GSH), whereas polysulfides are readily detected in garlic oil alone. Administration of garlic oil to cells rapidly increases intracellular polysulfide but has minimal effects on H₂S unless Cys or GSH are also present in the extracellular medium. We also observed that garlic oil and diallyltrisulfide (DATS) potently oxidized roGFP in buffer but did not affect DCF. This appears to be a direct polysulfide-mediated oxidation that does not require a reactive oxygen species intermediate. Conversely, when applied to cells, garlic oil became a significant intracellular reductant independent of extracellular Cys or GSH. This suggests that intracellular metabolism and further processing of the sulfur moieties are necessary to confer anti-oxidant properties to garlic oil in vivo.