Olive oil is a substrate, easy to be applied for detection of lipases. Tween 80 is a non-ionic surfactant and may be hydrolyzed by lipases. Members of the genus Rhizopus are one of the highest productive species for lipase on the market. During fermentation of agricultural byproducts, lipases and a variety of enzymes are produced. This study was carried out to detect the ability of Rhizopus azygosporus UICC 539 to use olive oil and Tween 80 at various temperatures. Agar block containing a concentration of R. azygosporus cells at approximately 106 cell/mL was inoculated on modified Czapek's Dox Agar (CDA) plates without a carbon source and with addition of 20 % Victoria Blue as an indicator. A combination of 0.5 % (v/v) olive oil and 0.5 % (v/v) Tween 80, or a combination of 1 % (v/v) olive oil and 1% (v/v) Tween 80 were added as carbon sources. Lipid hydrolysis was observed at day-3 and day-5 at various temperatures (30 °C, 35 °C, 40 °C, 45 °C and 50 °C). Clear zones were indicative of substrate hydrolysis. Enzymatic Index (EI) was determined by following the formula of: 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 olive oil and Tween 80 at all tested temperatures. R. azygosporus UICC 539 showed the highest EI at 2 % lipid substrate (1 % olive oil and 1 % Tween 80) after day-5 of incubation at 50°C. This study indicated that R. azygosporus UICC 539 was a potential candidate for fermentation processes of lipid-containing agricultural byproducts that requires higher temperatures.