The aim of this work was to investigate the effects of exposure to extremely low-frequency electromagnetic fields (ELF-EMF) both on biofilm formation and on mature biofilm of Helicobacter pylori. Bacterial cultures and 2-day-old biofilm of H. pylori ATCC 43629 were exposed to ELF-EMF (50 Hz frequency-1 mT intensity) for 2 days to assess their effect on the cell adhesion and on the mature biofilm detachment, respectively. All the exposed cultures and the respective sham exposed controls were studied for: the cell viability status, the cell morphological analysis, the biofilm mass measurement, the genotypic profile, and the luxS and amiA gene expression. The ELF-EMF acted on the bacterial population during the biofilm formation displaying significant differences in cell viability, as well as, in morphotypes measured by the prevalence of spiral forms (58.41%) in respect to the controls (33.14%), whereas, on mature biofilm, no significant differences were found when compared to the controls. The measurement of biofilm cell mass was significantly reduced in exposed cultures in both examined experimental conditions. No changes in DNA patterns were recorded, whereas a modulation in amiA gene expression was detected. An exposure to ELF-EMF of H. pylori biofilm induces phenotypic changes on adhering bacteria and decreases the cell adhesion unbalancing the bacterial population therefore reducing the H. pylori capability to protect itself.