This study investigated the microbial dynamics, physicochemical changes, nutritional profiles, antinutritional content, and probiotic potential of kombucha produced via two fermentation approaches: single fermentation of green tea extract and mixed fermentation involving green tea extract and pineapple juice using symbiotic culture of bacteria and yeast (SCOBY). Microbial enumeration and identification revealed a greater diversity of beneficial microorganisms, particularly Lactobacillus spp., Acetobacter spp., and fermentative yeasts in the mixed fermentation. Notably, mixed fermentation enhanced the presence of probiotic candidates such as Lactobacillus casei, L. plantarum, L. fermentum, and Saccharomyces spp. Physicochemical analysis showed a progressive decrease in pH and a corresponding increase in total titratable acidity, indicating active organic acid production. The final pH of mixed fermentation kombucha dropped to 2.99 by day 7 compared to 3.1 in the single fermentation. Proximate analysis indicated increased protein content (23.1 g/100g) and lower moisture content (78.3 g/100g) in mixed fermentation, suggesting better nutrient concentration and shelf stability. Mineral analysis demonstrated appreciable levels of potassium (5.8 mg/100g), magnesium (5.3 mg/100g), and iron (2.6 mg/100g) in the mixed-fermented product, surpassing the levels in the single fermentation counterpart. Antinutritional factors such as tannins, lectins, and trypsin inhibitors were significantly reduced post-fermentation, especially in the mixed fermentation sample, enhancing safety and nutritional quality. Probiotic characterization of seven bacterial isolates under simulated gastrointestinal conditions showed that most isolates exhibited bile salt tolerance and acid resistance, with these strains Lactobacillus casei, L. acidophilus, Pediococcus sp., and Streptococcus sp.) surviving all tested conditions, indicating strong probiotic potential. Sensory evaluation scores were higher for mixed-fermentation kombucha in all parameters, particularly taste (4.7), aroma (4.6), and appearance (4.8), confirming consumer acceptability. The findings suggest that the inclusion of pineapple juice in kombucha fermentation not only enhances microbial diversity and probiotic quality but also improves nutritional value, safety, and sensory appeal, offering a promising functional beverage in africa.