Tributyrin in solid media can be used to screen lipolytic activity. Lipolytic activity of many strains from Rhizopus has been described and some enzymes have been isolated. Members of Rhizopus play an important role in the decomposition of organic materials due to their effective extracellular enzyme production. Extracellular esterases have been produced by a member of Rhizopus. Esterases are used for degradation of domestic and agricultural biomass. There is always a demand for esterase enzymes with increased stability and selectivity for a variety of applications in industrial biotechnology. This study was carried out to detect the ability of Rhizopus azygosporus UICC 539 to use 1 % (v/v) and 2 % (v/v) tributyrin at various temperatures. Agar block (diameter 6 mm) containing a concentration of R. azygosporus cells at approximately 106 cell/mL was prepared from a 5-day old fungus in Potato Sucrose Agar (PSA) at 30 °C. The agar block was inoculated into 1 % (v/v) or 2 % (v/v) commercial tributyrin in Tributyrin agar base plates and incubated for 3 and 5 days at various temperatures (30 °C, 35 °C, 40 °C, 45 °C and 50 °C). Tributyrin agar base plates without the fungus served as control. Clear zones were indicative of tributyrin hydrolysis. The following formula was employed to calculate the Enzymatic Index (EI): R/r, where R was the diameter of the entire clear zone, and r was the diameter of the fungal colony. The results showed that R. azygosporus UICC 539 was able to use both tributyrin concentrations at all tested temperatures. High EI was detected at 50 °C at both tributyrin concentrations, with the highest EI at 1 % tributyrin after day-5 of incubation. In conclusion, R. azygosporus UICC 539 secreted esterolytic enzyme (esterase) into the medium when grown in 1 % and 2 % tributyrin. The strain utilized tributyrin in the temperature range of 30-50 °C. This study indicated that the enzymatic ability of R. azygosporus UICC 539 can be exploited in converting lipid-containing agricultural biomass in higher temperatures.