This study evaluated the impact of acerola by-product (ABP) on the viability and resistance to in vitro-simulated gastrointestinal (GI) conditions of Lactobacillus acidophilus LA-5 and Bifidobacterium longum BB-46, individually or in co-culture, in a fermented soy beverage (FSB) during 28 days of storage at 4 °C. Eight trials of FSB were studied, using a factorial 2³ design, in which the variables evaluated were Factor 1 (LA5), Factor 2 (BB46), and Factor 3 (ABP). The populations of microorganisms were determined using quantitative Real-Time PCR combined with propidium monoazide (PMA) treatment. Probiotic populations remained above 7.1 log CFU equivalent/mL throughout 28 days in the FSB, and the co-culture (LA5 + BB46) and the addition of ABP did not affect the viability of both probiotic strains (P > 0.05). However, ABP showed a positive effect on the survival of BB46 under in vitro GI conditions. The FSB supplemented with ABP revealed to be a good plant-based vehicle for the probiotics tested, mainly for BB46. Clinical trials are required to confirm the health benefits of the potential synbiotic soy-based product with ABP.