This study evaluated the protective effects of coproducts from agroindustrial processing of the tropical fruits acerola (Malpighia glabra L., ACE), cashew (Anacardium occidentale L., CAS), and guava (Psidium guayaba L., GUA) on the probiotics Lactobacillus paracasei L-10, Lactobacillus casei L-26, and Lactobacillus acidophilus LA-05 during freeze-drying and storage. The occurrence of damage to membrane integrity, membrane potential, and efflux activity of Lactobacillus cells after freeze-drying was evaluated by flow cytometry, and viable counts were measured immediately after freeze-drying and during 90 days of storage under refrigerated or room temperature conditions. Probiotic strains freeze-dried without substrate had the overall highest count reductions (0.5 ± 0.1 to 2.9 ± 0.3 log cycles) after freeze-drying. Probiotics freeze-dried with fruit processing coproducts had small cell subpopulations with damaged efflux activity and membrane potential. Average counts of probiotics freeze-dried with ACE, CAS, or GUA after 90 days of storage under refrigerated or room temperature were in the range of 4.2 ± 0.1 to 5.3 ± 0.2 and 2.6 ± 0.3 to 4.9 ± 0.2 log CFU/g, respectively, which were higher than those observed for strains freeze-dried without substrate. The greatest protective effects on freeze-dried probiotics were overall presented by ACE. These results revealed that ACE, CAS, and GUA can exert protective effects and increase the stability of probiotic lactobacilli during freeze-drying and storage, in addition to supporting a possible added-value destination for these agroindustrial coproducts as vehicles for probiotics and for the development of novel functional foods.