Herein, twelve fungal strains were isolated from a deteriorated historical manuscript dated back to the 18th century. The obtained fungal strains were identified, using the traditional method and ITS sequence analysis, asCladosporium herbarum(two strains),Aspergillus fumigatus(five strains),A. ustus(one strain),A. flavus(two strains),A. niger(one strain), andPenicillium chrysogenum(one strain). The ability of these fungal strains to degrade the main components of the paper was investigated by their activity to secrete extracellular enzymes including cellulase, amylase, gelatinase, and pectinase. The cell-free filtrate (CFF) ability of the probiotic bacterial strainLactobacillus rhamnosusATCC-7469 to inhibit fungal growth was investigated. The metabolic profile of CFF was detected by GC-MS analysis, which confirmed the low and high molecular weight of various active chemical compounds. The safe dose to be used for the biocontrol of fungal growth was selected by investigating the biocompatibility of CFF and two normal cell lines, Wi38 (normal lung tissue) and HFB4 (normal human skin melanocyte). Data showed that the CFF has a cytotoxic effect against the two normal cell lines at high concentrations, with IC50values of 525.2 ± 9.8 and 329.1 ± 4.2 µg mL−1for Wi38 and HFB4, respectively. The antifungal activity showed that the CFF has promising activity against all fungal strains in a concentration-dependent manner. The highest antifungal activity (100%) was recorded for a concentration of 300 µg mL−1with a zone of inhibition (ZOI) in the ranges of 21.3 ± 0.6 to 17.7 ± 0.5 mm. At a concentration of 100 µg mL−1, the activity of CFF remained effective against all fungal strains (100%), but its effectiveness decreased to only inhibit the growth of eight strains (66%) out of the total at 50 µg mL−1. In general, probiotic bacterial strains containing CFF are safe and can be considered as a potential option for inhibiting the growth of various fungal strains. It is recommended that they be used in the preservation of degraded historical papers.