Lichen species have unique culture media preferences, and established cultures are known for the synthesis of secondary metabolites. This paper reports observations on the developmental stages and secondary compound biosynthesis by the mycobiont and whole thallus cultures of Buellia subsororioides. It also investigates the suitable media compositions for the culture growth, the role (nutrient or stressor) of sucrose concentrations on the growth stages, biomass, secondary compound profiles, and the quantity of biosynthesized known compounds/g of culture biomass for each treatment using mycobiont cultures. The ascospore-derived mycobiont cultures and thallus macerate-derived whole thallus cultures of B. subsororioides were established and grown using malt yeast extract (MY) medium. Mycobiont cultures were subcultured in MY medium supplemented with sucrose and its concentrations ranging from 0 to 30 % (with 2 % increment between treatments) for 120 days. The molecular identity of cultures was confirmed using nuclear ribosomal Internal transcribed spacer (ITS) DNA sequences obtained from the cultured mycobiont and from the natural thallus. The ITS DNA sequences of the mycobiont showed 99 % similarity with the sequences of the natural thallus. The mycobiont cultures under varying sucrose concentrations initiated as white cottony stages and transformed to brown compact mycelia, with optimum biomass and biosynthesis of nine secondary compounds in MY 10 %. The number of compounds (1–9) varies according to treatments. The whole thallus cultures (MY 0 %) showed a profile of secondary compounds similar to that of the natural thalli along with a trace of one unknown compound. The obtained results are encouraging for the synthesis of the desired quantities of lichen secondary compounds through cultures for relevant applications.