Vinasse is a distillery effluent which is produced during sugarcane molasses-based ethanol fermentation. Although numerous researches have been conducted on potential applications of vinasse, it’s still underutilized in practice. This study evaluated the potential use of vinasse in preparing growth media for ethanologenic yeast and cellulolytic fungi that are used in ethanol production from sugarcane bagasse. Vinasse agar was prepared with two vinasse concentrations (50% & 100% v/v). Two Saccharomyces isolates (Y4 and Ysev) were serially diluted in sterile saline (0.09%) and the 10-5 dilution was plated in triplicate, while yeast extract peptone dextrose (YEPD) agar was the positive control. The cultures were aerobically incubated at 37 ˚C for 24 hours. 100 µl of Trichoderma viridae spore suspension (1×108) was inoculated in to a series of vinasse solutions (0% to 100%, 30 ml) containing sugarcane bagasse (1.0 g) as the carbon source. After one week of incubation at 30 ˚C with 120 rpm shaking, the total cellulase activity of crude enzyme extracts were determined. Park’s cellulase production medium was the positive control. In vinasse agar, the highest growth of 1.13×104 CFU/ml and 1.00×104 CFU/ml were observed in Ysev-cultured, 100% and 50% vinasse agar media respectively. Its growth in YEPD agar (1.05 ×104CFU/ ml) wasn’t significantly different from values at 100% and 50% vinasse. However, Y4’s growth in vinasse agar was significantly lower than Ysev. In 50 % vinasse agar, 8.33×103CFU/ml of Y4 was observed which was not significantly different from 8.6×103 CFU/ml growth in YEPD agar. The lowest growth was observed in Y4 as 4.87×103 CFU/ml in 100% vinasse agar. The growth in vinasse was isolate-dependent. The highest total cellulase activity of T. viridae was observed at 50% vinasse as 1.46 FPU/ml whereas in Park’s medium it was only 0.859 FPU/ml. These results show that vinasse has facilitated the growth of yeast isolates and T. viridae which proves the potential application of vinasse in formulating low-cost culture media for fungi.