Indole is produced when Escherichia Coli K12 is subjected to stress in a long duration mixed culture with Bacillus subtilis 168. Due to the therapeutic uses of Indole, a mathematical model of the mixed culture process underlying the growth and providing the optimal operating condition could prove immensely useful for the large scale production of Indole in industries. In this work, an experimental study has been conducted to study the long duration mixed culture effect on Escherichia Coli and Bacillus Subtilis in minimal media. Experimental investigation of individual growth profile of both the bacteria before and after the mixed culture is also conducted with glucose as substrate to analyze the effect of interaction. A computational strategy for identification of optimal unstructured model for mixed culture using hybrid optimization technique has been adopted. For modelling the mixed culture, dynamics of long duration passage experiment dynamic optimization technique has been implemented, which can be used to find optimal operating conditions. This work represents the first instance, where hybrid and dynamic optimization techniques have been applied for unstructured modeling and optimization of mixed culture, acting as a novel framework for optimal production of Indole at industrial scale and drug discovery. © 2019 IEEE.