Organosulfur compounds (OSCs) are secondary metabolites produced by different Allium species which present important biological activities such as antimicrobial, antioxidant, anti-inflammatory antidiabetic, anticarcinogenic, antispasmodic, etc. In recent years, their use has been promoted in the agri-food industry as a substitute for synthetic preservatives, increasing potential accumulative exposure to consumers. Before their application in the food industry, it is necessary to pass a safety assessment as specified by the European Food Safety Authority (EFSA). This work reviews the scientific literature on OSCs regarding their in vitro toxicity evaluation following PRISMA guidelines for systematic reviews. Four electronic research databases were searched (Web of Science, Scopus, Science Database and PubMed) and a total of 43 works were selected according to predeterminate inclusion and exclusion criteria. Different data items and the risk of bias for each study were included. Currently, there are very few in vitro studies focused on investigating the potential toxicity of OSCs. Most research studies aimed to evaluate the cytotoxicity of OSCs to elucidate their antiproliferative effects focusing on their therapeutic aspects using cancer cell lines as the main experimental model. The results showed that diallyl disulfide (DADS) is the compound most studied, followed by diallyl trisulfide (DATS), diallyl sulfide (DAS), Allicin and Ajoene. Only 4 studies have been performed specifically to explore the safety of OSCs for agri-food applications, and genotoxicity studies are limited. More toxicity studies of OSCs are necessary to ensure consumers safety and should mainly be focused on the evaluation of genotoxicity and long-term toxicity effects.