This study aims at an in vitro characterization of the acid and bile tolerance of Lactobacillus fermentum InaCC B1295 (LFB1295) encapsulated with hydrogel cellulose microfibers (CMF) from oil palm empty fruit bunches (OPEFBs). The viability at different storage temperatures was assessed. The experimental design used in this research was an in vitro trial. The microencapsulated probiotic was stored at 25 °C and 4 °C for 28 days. LFB1295 encapsulated with cellulose microfiber hydrogel from OPEFB showed a stable viability of probiotic bacteria at pH 2 and 0.5% (m/v) oxgall. In addition, the microencapsulation maintained the viability at 25 °C and 4 °C at 0, 14, and 28 days. The characterization of the encapsulant CMF-OPEFB showed that the thickness of CMF was in the range of 5–15 μm, and XRD patterns showed that CMF was of the cellulose I type with a crystallinity index of 77.08%. Based on its resistance to hydrogen peroxide, ability to scavenge DPPH radicals, and activity in scavenging hydroxyl radicals, LFB1295 encapsulated with CMF hydrogel of OPEFB exhibits antioxidant properties as good as the scavenging ability of DPPH radicals with IC50 of 36.880, 188.530, and 195.358 µg/mL, respectively, during storage for 0, 14, and 28 days at room and refrigerated temperature. Furthermore, hydroxyl radicals (HR)-scavenging activity showed an increased inhibition along with the increasing concentration of the Fenton reaction and decreasing concentration of cell-free supernatant (CFS) during storage time. In vitro safety tests, including hemolytic activity, biogenic amines, cytolysin, and gelatinase production, showed that the encapsulated LFB1295 was safe to use as a probiotic. The results of the inhibitory activity against hydrogen peroxide LFB1295 show that the higher the concentration of H2O2, the lower the inhibition value during 28 days of storage. Based on the storage temperature, the inhibition of LAB against H2O2 based on different storage temperatures showed a better level of the inhibition at cold temperatures compared to at room temperature.