Bacteria inhabiting the gut of malaria vectors can strongly influence their biology and competence to transmit Plasmodium parasites and other pathogens. Understanding the composition of the culturable gut microbiota of Anopheles mosquitoes, together with the factors shaping its community structure is of relevance to implement alternative vector control approaches, such as paratransgenesis. This study aimed to isolate and characterize the culturable bacterial microbiota present in the gut of two main Colombian malaria vectors and to determine how some factors such as mosquito species, geography, and feeding status can modulate their community assembly. For this, Anopheles darlingi and An. nuneztovari were collected in two malaria-endemic regions under two feedings status. Bacterial isolates from homogenized mosquito guts were morphologically and biochemically characterized and their taxonomy was identified via sequencing of the 16S rRNA gene. Results showed that gram-negative bacilli, particularly of the Proteobacteria phylum, are predominant in the gut of the two vector species, regardless of geography and feeding status. At the genus level, Enterobacter, Acinetobacter and Bacillus were common among the two vector species and geographic sites, however, there were genera that were locality and vector specific. Furthermore, the presence of blood in the mosquito gut resulted in a negative impact on bacterial richness at the genus level. The present study showed that the culturable gut bacterial microbiota of two Colombian malaria vectors vary according to geography and feeding status. Additionally, the use of traditional culture-based techniques allowed the generation of a bacterial culture collection to further investigate their role as potential biocontrol agents.