This study aimed to investigate controlled fermentation of cocoa beans with selected yeasts as starter cultures via integrating microbiological, biochemical, and chromatographic analyses. The steps involved in the yeast starter culture test were of the following order: 1) counting, isolation, purification, and biochemical identification of yeasts, 2) selection of ethanol-producing yeasts, 3) selection of thermotolerant yeasts, and 4) evaluation of physicochemical parameters of the selected yeasts in controlled fermentation of cocoa (F1 -Saccharomyces ssp. and Hanseniaspora ssp. and F2 -spontaneous fermentation -control). A total of 32 yeasts were isolated from three sampling points (M1, M2, and M3), which comprised 50% Candida ssp., 9.4% Rhodotorula ssp., 18.8% Saccharomyces ssp., and 18.8% Hanseniaspora ssp. The yeasts identified as Saccharomyces ssp. (n = 6) were subjected to the ethanol production test. Saccharomyces spp. CLV09 showed the highest concentration of ethanol in the simulated cocoa medium (3.5% v/v). Hanseniaspora spp. CVL20 and CVL19 strains showed the highest thermotolerance at 42EC after 72 h of growth. The starter cultures with Saccharomyces ssp. and Hanseniaspora ssp. showed a similar growth rate of the mesophilic aerobic population in both F1 and F2. Fermentation of the starter culture showed a higher production of organic acids than spontaneous fermentation (F2). Thus, Saccharomyces ssp. and Hanseniaspora ssp. can be used as a starter culture in cocoa fermentation.