The purpose of the present study was to evaluate yellow mombin (Spondias mombin L.) juice as a vehicle for the Lactobacillus acidophilus NRRL B-4495 probiotic. The initial pH and fermentation temperature conditions were optimized by central composite rotational design. The beverage was evaluated for its chemical composition, bioactive properties, microbiological stability, survival in simulated gastrointestinal conditions and sensory analysis. The ideal conditions for probiotic juice production were an initial pH of 6.4 and 16 h of fermentation, with maximum viability of 12.9 ± 0.4 Log CFU/mL. The fermented juice showed a total phenolic concentration of 94.90 ± 7.12 GAE/mL and antioxidant activity, as measured by DPPH (0.31 ± 0.00 μmol TE/mL) and ABTS sequestration (2.59 ± 0.30 μmol TE/mL). Antibacterial activity could also be observed against S. aureus, E. coli and K. pneumoniae. The obtained formulation showed good microbiological stability when stored at 4ºC for 28 days. In addition, there was no significant change in viability after exposure to simulated gastrointestinal conditions. The sensory analysis showed that the probiotic beverage was not well accepted. However, the Just-About-Right (JAR) ideal scale test enabled identifying the specific attributes which need to be improved from the tasters' point of view so that it is possible to improve product acceptance.