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This study aimed to describe the microbial populations involved in the production of traditional raw milk, semi-dry cheese from Honduras, Central America, particularly the indigenous lactic acid bacteria (LAB) responsible for the organoleptic characteristics of this product. A total of 48 cheese samples were collected from seven of the 18 country’s Departments. Phenotypic methods were used to count, identify and compare the composition of bacteria and yeasts populations in the samples. Twenty LAB isolates were randomly selected to analyse its 16S-23S rRNA spacer region. This method was also applied to understand the diversity of bacterial communities present in the samples. LAB colonies reached average counts of 4.79 lg CFU g-1. Twelve LAB species were identified based on a conventional phenotypic approach. Lactococcus lactis and Enterococcus faecium were the most frequently identified species according to microbiological and molecular methods. No significant correlation was observed among LAB species and geographical distribution. Four genera and five species were identified among the isolated yeasts. This is the first report describing the indigenous biota of traditional raw milk cheeses from Honduras. Our results may provide useful information for valuable strain selection as starter cultures for the industrialization of traditional dairy products in Honduras.