Coffee fermentation represents a natural bioreactor in which microbial metabolism shapes the aroma, acidity, and sensory identity of one of the most valued beverages in the world. This study optimized a semi-controlled fermentation process using commercial lactic acid bacteria (LAB) and yeasts under wet conditions to enhance the sensory quality of Coffea arabica var. Colombia at a smallholder scale. A sequential experimental strategy combined a definitive screening design (DSD) to identify key variables—fermentation time (X₁), inoculum concentration (X₂), water content (X₃), microorganism type (X₄), and inoculum preparation method (X₅)—with a factorial optimization step. Relative microbial growth was included as a covariate, and the sensory score (S) of the final coffee served as the main response variable, as determined by a certified Q-grader following the Specialty Coffee Association (SCA) cupping protocol. During fermentation, pH, titratable acidity, soluble solids, and microbial viability were monitored to characterize process dynamics. The DSD revealed that microorganism type, fermentation time, and inoculum preparation method were the most influential factors on sensory outcomes. Optimization predicted the highest S value (85.14) with a yeast inoculum prepared from fresh commercial Saccharomyces cerevisiae yeast (Levapan, Colombia) and a fermentation duration of 48 h, using minimal inoculum concentration (3%) and water content (50%). Experimental validation confirmed these conditions, achieving a relative error of 0.36%. The results demonstrate that accessible commercial microbial inoculum can significantly improve process control, microbial performance, and sensory quality without requiring specialized infrastructure. This biotechnological approach provides a cost-effective and scalable fermentation model for smallholder producers, integrating microbial optimization with sustainable practices to strengthen participation in the specialty coffee market.