The demand for food of animal origin is expected to increase by 2050. Since traditional protein sources for monogastric diets are failing to meet the increasing demand for additional feed production, there is an urgent need to find alternative protein sources. The larvae of Hermetia illucens emerge as efficient converters of low-quality biomass into nutritionally valuable proteins. Many studies have been performed to optimize H. illucens mass rearing on a number of organic substrates and to quantitatively and qualitatively maximize the biomass yield. On the contrary, although the insect microbiota can be fundamental for bioconversion processes and its characterization is mandatory also for safety aspects, this topic is largely overlooked. Here, we provide an in-depth study of the microbiota of H. illucens larval midgut, taking into account pivotal aspects, such as the midgut spatial and functional regionalization, as well as microbiota and nutrient composition of the feeding substrate.