Non-toxigenic Vibrio cholerae (NVC) isolates are associated with diarrheal disease globally. NVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. This study aims to give a better picture of the evolution of the incidence of NVC in Spain from 2012 to 2022. In this context, we realized a time-series and a descriptive analysis for the cases submitted to the National Center of Microbiology (NCM) during this period. In addition, we conducted whole genome sequencing (WGS) of a selection of isolates to elucidate the heterogeneity of the sporadic clinical strains of NVC among patients residing in Spain. First, we observed an increase in the number of isolates sent to the NCM after 2019 which was not associated to a change in the national surveillance protocol. Risks of infection and hospitalization increased with the age of the case. Second, we found a high diversity of NVC strains, and we showed that the usefulness of WGS studies is limited in outbreak situation to find the infectious source. Finally, we characterized the genetic determinants responsible for antimicrobial resistance and virulence and found that 21% of the isolates were resistant to beta-lactamases. The present study is the first in Spain to report data on non-toxigenic cases at the national level. Because of the high percentage of hospitalization observed for NVC cases, it might be beneficial to reinforce the surveillance of NVC in Spain and testing for V. cholerae in all the suspected cases.