Brewers, seeking distinctive flavor profiles, have been captivated by koji molds, deeply embedded in oriental culinary traditions, due to their intricate enzymatic profiles. This study investigates temperature and fermentation time effects on the malt-processing enzymes (amylases, cellulases, and proteases) from Aspergillus oryzae and Aspergillus luchuensis koji molds. Pearled barley, following steeping, cooking, and cooling, underwent fermentation with koji molds at 31 °C and 34 °C for up to 42 h. Enzymes were extracted from fermented barley by suspending it in a pH 5 buffer, agitating it in a water bath at 30 °C for 30 min, and separating the supernatant through centrifugation. This supernatant was utilized for enzymatic profiles assessment. Evaluated at 50 °C, enzyme activities included proteolytic activity using azocasein at pH 8, cellulolytic activity via DNS using carboxymethylcellulose at pH 5, and amylolytic activity using soluble starch at pH 6, assessed through dextrinization via iodine staining and saccharification via DNS. A. oryzae demonstrated superior overall enzymatic activity, particularly excelling in proteolytic and saccharifying activities, reaching peaks of 2.9 and 73.4 U/mL, respectively. In contrast, A. luchuensis exhibited lower activity peaks at 0.8 and 19.1 U/mL. However, its dextrinizing activity closely paralleled that of A. oryzae. Overall, enzymatic activities exhibited an upward trend with increasing temperature and fermentation time. Nevertheless, cultivating of A. oryzae at 31 °C favored enhanced cellulolytic activity at 42 h (8.2 U/mL compared to 4.9 U/mL at 34 °C) and increased proteolytic activity beyond the 32-hour mark. In conclusion, this study underscores the significant impact of temperature and fermentation time on the enzymatic profiles of A. oryzae and A. luchuensis koji molds. Consequently, further investigation is warranted to explore the effects of these factors on the potential applications of koji molds in food, including flavor development and malt processing in brewing.