This study aimed to relate the acquisition of different antibiotic resistances and the corresponding physiological responses to cold stress of Lactobacillus delbrueckii subsp. bulgaricus strain CFL1. Six resistant mutants were spontaneously obtained and studied depending on the target of the antibiotic: (i) bacitracin and vancomycin (Bac(R), Van(R), wall synthesis), (ii) novobiocin (Nov(R), DNA replication), and (iii) kanamycin, spiramycin, streptomycin (Kan(R), Spi(R), Str(R), RNA translation). The mutations modified the growth and the cold stress response at three different physiological levels: (i) Van(R) and Spi(R) mutants showed significant lower growth rates compared to the wild type strain. (ii) Van(R) and Bac(R) mutants displayed a slightly higher resistance to a freezing-thawing challenge whereas Str(R) and Spi(R) mutants were more sensitive compared to the wild type. (iii) The recovery of acidification activity after freezing and during frozen storage was improved by considering the Nov(R) strain, but not with the Van(R) and Spi(R) mutants. Thus, acquisition of some antibiotic resistance by spontaneous mutation led to modification of the cold stress response. The hypothesis of a unique cellular thermostat is discussed regarding the diversity of the tested antibiotics.