Many studies have been focusing on the production of microbial lipid to generate biodiesel from sterile wastewater while removing part of organic matters. Our previous study showed that lipids could be produced from non-sterile distillery wastewater using the increased initial cell density of oleaginous yeast Rhodosporidium toruloides while generating lots of spent seed culture medium. This study explores the reuse potential of spent seed culture medium while saving resource and cost. When the medium was used for the 2nd and 3rd times without addition of extra nutrient, the biomass produced was around 3.17 and 2.65 g/L respectively. The cells produced in the 2nd time reused medium without extra nutrient showed similar performance to the fresh mediumin lipid production (biomass 7.42±0.31 g/L, lipid yield 2.74±0.42 g/L and lipid content 36.90±4.36%) and removal efficiencies for organics and nutrients (chemical oxygen demand, total nitrogen and total phosphorus removal of 86.47±1.40%, 50.73±6.15% and 74.36±3.39% respectively) in non-sterile distillery and domestic mixed wastewater (1:1, v/v) after two-day cultivation. Around 30% reduction in the material cost for the medium preparation could be achieved by the spent medium reuse.