Food is a principal need for the continued survival of all living organisms. Food safety is an essential demand that must be achieved to avoid its refusal. The contamination of food by the toxigenic fungi threatens food safety and security. This review aims to get an overview of the pathway of AFB1 enzyme biosynthesis and their interaction with the genes relating to the cluster genes of AFB1. It also aims to demonstrate the impact of the global environmental conditions that influence the secretion process of aflatoxin and the current information indicating that genes regulated by such environmental signals are interconnected with aflatoxin biosynthesis. Various fungi exist in food commodities, but not all can secret mycotoxins. Aspergillus fungi species are one of the dangerous enemies that have caused food condemnation. Aspergillus strains mainly produce aflatoxins as a secondary metabolite during their bioactivities. Aflatoxins are classified by the International Agency for Cancer Research, where aflatoxin B1 (AFB1) is considered a class I carcinogen. Aspergillus fungi may biosynthesis the aflatoxin through about 30 genes, with principal ones including aflR, ver-1, verA, avfA, and nadA. These genes cover about 75 kb of the fungal genome. With the advancement of molecular tools, the research of filamentous fungi developed dramatically and offered valuable opportunities to explain specific fungal pathways, such as the formation of secondary metabolites. Using the advantage of these technologies to enhance food safety and security, the molecular analysis of toxigenic fungi will hopefully understand the role influencing the formation of toxins and allow the creation of new successful fungal toxicity control strategies. Extensive research has been done on the genes that are implicated in the formation of AFB1, which is one of the most harmful human and animal carcinogenic toxins. The existing review discusses the responsibilities of these genes and their potential effect on forming AFB1. The emphasis was on the Aspergillus flavus and Aspergillus parasiticus, deemed the critical pathogens characterized as the significant AFB1-generator in crops. This review perfectly understands factors, related genes, mechanisms, and pathways of aflatoxin production as a critical hazard that threatens food safety production. This review provided complete knowledge about reducing aflatoxin in the food chain supply. It supported the idea of eliminating aflatoxin secretion using novel strategies, leading to more safety in food production.