S-allyl-L-cysteine (SAC) is a sulfur compound present in fresh garlic. The reference literature describes its anticancer, antioxidant and neuroprotective effects. Breast cancer is infamously known as one of the most commonly diagnosed malignancies among women worldwide. Its morbidity and mortality make it reasonable to complete and expand knowledge on this cancer’s characteristics. Hydrogen sulfide (HS) and its naturally occurring donors are well-known investigation subjects for diverse therapeutic purposes. This study was conducted to investigate the SAC antiproliferative potential and effect on three enzymes involved in HS metabolism: 3-mercaptopyruvate sulfurtransferase (MPST), cystathionine γ-lyase (CTH), and cystathionine β-synthase (CBS). We chose the in vitro cellular model of human breast adenocarcinomas: MCF-7 and MDA-MB-231. The expression of enzymes after 2, 4, 6, 8, and 24 h incubation with 2.24 mM, 3.37 mM, and 4.50 mM SAC concentrations was examined. The number of living cells was determined by the MTS assay. Changes in cellular plasma membrane integrity were measured by the LDH test. Expression changes at the protein level were analyzed using Western blot. A significant decrease in viable cells was registered for MCF-7 cells after all incubation times upon 4.50 mM SAC exposure, and after 6 and 24 h only in MDA-MB-231 upon 4.50 mM SAC. In both cell lines, the MPST gene expression significantly increased after the 24 h incubation with 4.50 mM SAC. S-allyl-L-cysteine had opposite effects on changes in CTH and CBS expression in both cell lines. In our research model, we confirmed the antiproliferative potential of SAC and concluded that our studies provided current information about the increase in MPST gene expression mediated by S-allyl-L-cysteine in the adenocarcinoma in vitro cellular model for the MCF-7 and MDA-MB-231 cell lines. Further investigation of this in vitro model can bring useful information regarding sulfur enzyme metabolism of breast adenocarcinoma and regulating its activity and expression (gene silencing) in anticancer therapy.