The Tenebrio molitor insect flour (TMIF) is considered a nutritive food ingredient, but its impact at the gut microbiota level and its potential prebiotic activity still need be assessed. For such studies, an in vitro simulation model of gut microbiota was optimized. Pure cultures of Lactobacillus and Bifidobacterium strains in monocultures and co-cultures (pairs and consortium) were used in this model to evaluate the effect of TMIF on the viability and metabolic activity of those bacteria. The optimization of the in vitro model of the gut microbiota was successful, and growth of the most important groups of bacteria in the gut microbiota was observed. So this model can be used to study the effects of other ingredients at that level. It also enabled pinpointing the prebiotic effects of the studied TMIF suggesting possible symbiotic interactions. In addition, the direct effect of the TMIF on bacterial cells, when in nutritive stress was also evaluated. In terms of TMIF effects on probiotic bacteria viability and growth, no negative effects were observed, and even an enhancement of growth and an increase of production of short chain fatty acids (SCFA) and lactate, in most of the cases, was observed. Also, this study showed that TMIF helps in maintaining the viability of bacteria during incubation time when these are under nutritional stress conditions. A potential prebiotic effect of TMIF is then predictable with this study, opening a path to new research on this subject.