Two fermentations in skim milk were used to evaluate the effectiveness of the bacteriocin Thermophilin-T produced “in situ” by Streptococcus thermophilus ACA-DC 0040 in inhibiting spore outgrowth of Clostridium sporogenes ACA-DC 3888 under conditions simulating Graviera cheese manufacture and ripening. In the experimental fermentation, S. thermophilus ACA-DC 0040 and Clostridium spores were used. In the control fermentation, a non-bacteriocin-producing S. thermophilus ACA-DC 004 strain and Clostridium spores were used. The temperature program used and the pH changes were similar to those observed in Graviera cheese production and ripening. Clostridium spore counts and organic acids were measured during both fermentations. In the experimental trial, bacteriocin production by S. thermophilus ACA-DC 0040 reduced Clostridium spores by 1.79 log units during the ripening phase. Conversely, the Clostridium spore count significantly increased in the control trial from the 15th day onward by about 2.9 log units and showed elevated levels of acetic, propionic, and butyric acids, along with decreased lactic acid, which is clearly linked to the “Late Blow Defect” profile. The results of our study on the inhibition of C. sporogenes spore outgrowth provide useful information for establishing an effective biological control system, in addition to other barriers used during Graviera cheese manufacturing.