Human tuberculosis (TB) caused by Mycobacterium bovis surveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused by M. bovis over a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles of M. bovis strains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused by M. bovis was 1·76%, significantly associated to extra-pulmonary localization (P = 0·004) and to being elderly (P < 0·001) and Italy-born (P = 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that most M. bovis strains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases of Mycobacterium bovis BCG infection were also characterized. In conclusion, the rate of human TB caused by M. bovis was not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.