Background/Objectives: The influenza vaccination of healthcare workers (HCWs) is recommended each autumn and winter season by the relevant authorities in EU/EEA countries. The objective of this study was to evaluate the impact of repeated trivalent influenza vaccine (TIV) uptake during the 2017/2018 and 2018/2019 seasons on vaccine-derived immunity against influenza. Methods: A cohort study of HCWs vaccinated with an annual TIV was conducted from October 2017 to June 2019. The protective antibodies against the influenza vaccine strains were assessed at three time points: prior to vaccination and at one and six months following vaccination for each season. Sera were tested by hemagglutination inhibition assay. Participants were grouped according to their history of TIV vaccination over four seasons (since 2015/16), with the groups designated as “frequently vaccinated” (≥3 vaccines) and “occasionally vaccinated” (≤2 vaccines). Seroprevalence, geometric mean titer (GMT) and seroconversion rate were compared between the frequently and occasionally vaccinated groups. Results: A total of 97 healthcare workers (HCWs) were enrolled in the study; 49 HCWs participated in both seasons. Thirty-two (43.2%) and forty-three (59.7%) individuals had ≥3 vaccines since 2015/2016, at recruitment and during the 2017/2018 and 2018/2019 influenza seasons, respectively. One month following vaccination, HCWs who had received occasional vaccinations demonstrated a higher prevalence of protective antibodies and a greater GMT for both influenza A(H1N1)pdm09 and A(H3N2) viruses. For influenza B Victoria, the frequently vaccinated HCWs demonstrated a higher seroprevalence rate, seroconversion, and GMT. Conclusions: Previous vaccination can influence the immune response, although without substantially compromising the immunogenicity of annual influenza vaccination. HCW annual influenza vaccination is required to re-establish and maintain the antibody titers against influenza.