Various chemical modulators can be used to directly regulate the lipid biosynthesis pathway, with similar effects to gene overexpression and interference approaches. Display omitted •Chemicals can be used to improve the precursor supply and block competing pathways.•The cell permeability and oxidative stress can be regulated by chemicals.•Chemicals is a valuable way for regulating the large-scale cultivation of algae.•Many potential effective chemicals were put forward. Microalgae have attracted great attention as a promising sustainable resource for biofuel production. In studies aiming to improve lipid accumulation, many key enzymes involved in lipid biosynthesis were identified and confirmed, but genetic engineering remains a challenge in most species of microalgae. In an alternative approach, various chemical modulators can be used to directly regulate the lipid biosynthesis pathway, with similar effects to gene overexpression and interference approaches, including improving the precursor supply and blocking competing pathways. The produced lipid can be protected from being converted into other metabolites by the chemicals such as lipase inhibitors. In addition, a few chemicals were also demonstrated to greatly influence cell growth and lipid accumulation by indirect regulation of the lipid biosynthesis pathway, such as increasing cell permeability or regulating oxidative stress. Thus, adding chemical modulators can be a useful alternative strategy for improving lipid accumulation in large-scale cultivation of microalgae.