Banana puree, due to its nutritional composition, is a good substrate for fermentation in the development of probiotic products. The production of banana puree mainly consists of three phases, i.e., raw material pretreatment, heat treatment, and the addition of anti-browning agents. In this study, we conducted three experimental protocols to evaluate the effect of ripeness grade, heat treatment, and ascorbic acid addition on fermentation performance. At the end of each protocol, the substrate was subjected to the fermentation process (37 °C, 48 h), and then measurements of pH reduction, microbial growth, and lactic acid production were used as markers in the analysis of fermentation performance. Ripe bananas produced better results than unripe bananas whose fermentation appeared to be inhibited. Therefore, ripe bananas were used to test the effect of two different heat treatments (sterilization (121 °C, 20 min) versus tyndallization (70 °C, 30 min; 37 °C, 30 min; 70 °C, 30 min)) on banana puree fermentation, and no significant differences were observed. Finally, 500 or 1000 ppm of ascorbic acid, normally used as an anti-browning agent, was added to ripe tyndallized bananas. No differences in fermentation results were observed between the two tested conditions, though values obtained for growth and lactic acid production were significantly lower than those from fermentation of banana puree without ascorbic acid.