This study focuses on the biodegradation of difluorobenzenes (DFBs), compounds commonly used as intermediates in the industrial synthesis of various pharmaceutical and agricultural chemicals. A previously isolated microbial strain (strain F11), identified as Labrys portucalensis, able to degrade fluorobenzene (FB) as sole carbon and energy source, was tested for its capability to degrade 1,2-, 1,3- and 1,4-DFB in batch cultures. Strain F11 could use 1,3-DFB as a sole carbon and energy source, with quantitative release of fluoride, but 1,4-DFB was only degraded and defluorinated when FB was supplied simultaneously. Growth of strain F11 with 0.5 mM of 1,3-DFB led to stoichiometric release of fluoride ion. The same result was obtained in cultures fed with 1 mM of 1,3-DFB or 0.5 mM of 1,4-DFB, in the presence of 1 mM of FB. No growth occurred with 1,2-DFB as substrate, and degradation of FB was inhibited when supplied simultaneously with 1,2-DFB. To our knowledge, this is the first time biodegradation of 1,3-DFB as a sole carbon and energy source, and cometabolic degradation of 1,4-DFB, by a single bacterium, is reported.