Introduction: Vineyard is a crop where a large number of pesticides are applied; exposure to pesticides may occur in farmers and the general population living close to the treated area. This work aimed to investigate hair as a matrix for the assessment of cumulative and aggregate exposure to pesticides in potentially exposed individuals. Methods: Twenty agricultural workers (AW), 4 agricultural worker relatives (AR), and 5 research staff members (RS) were involved in the study. Hair samples were collected before and after the application season (PRE- and POST-EXP samples) to obtain 18 paired samples. Records with the name and the quantity of applied pesticides were obtained; twenty-seven pesticides were measured in hair by solvent extraction and LC-MS/MS. Results: During the study season, AW applied 14 different pesticides with median amount ranging from 12 to 7200 g. The most popular pesticides were dimethomorph, penconazole, cyazofamid, fenamidone and quinoxyfen, applied from 94 to 69% of AW. In AW, in PRE-EXP samples the majority of used pesticides was detectable (with detection rates from 6 to 88%), with median concentrations of few pg/mg hair; in the POST-EXP samples the frequency of detected values increased (from 25 to 100%), with median concentrations up to two orders of magnitude higher. In AR, most pesticides were quantifiable only in POST-EXP samples and with lower concentration in comparison with AW; in RS, in both PRE- and POST-EXP samples only a few pesticides were quantifiable with very low levels. In AW, a linear correlation (r = 0.682 on log-transformed data, p < 0.01) was found between the total amounts of applied pesticides during the season and their concentration in hair. Conclusion: The study shows that the majority of assessed pesticides was incorporated into hair of AW and AR. The increased frequency of detection and level at the end of the season and the correlation between pesticide in hair and the amount of applied pesticides, reinforce the use of hair for quantitative biomonitoring of cumulative exposure to pesticides.