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Background: Due to potentially immune-escaping virus variants and waning immunity, a third SARS-CoV-2 vaccination dose is increasingly recommended. However, data in patients with cancer are limited., Patients and Methods: We measured anti-SARS-CoV-2 spike protein antibody levels after the third vaccination dose in 439 patients with cancer and 41 health care workers (HCW) at an academic centre in Austria and a rural community hospital in Italy. Adverse events were retrieved from questionnaires., Results: Overall, 439 patients and 41 HCW were included. SARS-CoV-2 infections were observed in 62/439 (14.1%) patients before vaccination and in 5/439 (1.1%) patients after ≥1 dose. Longitudinal analysis revealed a decrease of antibody levels between 3 and 6 months after second vaccination in patients with solid tumours (p < 0.001) and haematological malignancies without anti-B cell therapies (p < 0.001). After the third dose, anti-S levels increased compared to the first/second dose. Patients receiving B cell-targeted agents had lower antibody levels than patients with haematological malignancies undergoing other treatments (p < 0.001) or patients with solid tumours (p < 0.001). Moreover, anti-S levels correlated with CD19+ (B cell) and CD56+ (NK cell) counts in peripheral blood. The most frequent adverse events after the third dose were local pain (75/160, 46.9%), fatigue (25/160, 15.6%) and fever/chills (16/160, 10.0%). Patients with cancer had lower anti-S levels than HCW (p = 0.015)., Conclusions: This study in patients with cancer shows improved antibody levels after the third vaccination dose at an acceptable side-effect profile. Lower antibody levels than in controls underline the need for further follow-up studies and dedicated trials., Competing Interests: Conflict of interest statement Anna Sophie Berghoff has received research support from Daiichi Sankyo, Roche, and honoraria for lectures, consultation or advisory board participation from Roche, Bristol-Meyers Squibb, Merck, Daiichi Sankyo as well as travel support from Roche, Amgen and AbbVie. Thorsten Fuereder has received honoraria for lectures, consultation or advisory board participation from the following for-profit companies: MSD, Merck Darmstadt, Roche, Bristol-Myers Squibb, Accord, Sanofi, Boehringer Ingelheim as well as travel support from Roche, MSD and Bristol-Myers Squibb. The following for-profit companies have supported clinical trials and contracted research conducted by TF with payments made to his institution: MSD, Merck Darmstadt, Bristol-Myers Squibb. Matthias Preusser has received honoraria for lectures, consultation or advisory board participation from the following for-profit companies: Bayer, Bristol-Myers Squibb, Novartis, Gerson Lehrman Group (GLG), CMC Contrast, GlaxoSmithKline, Mundipharma, Roche, BMJ Journals, MedMedia, Astra Zeneca, AbbVie, Lilly, Medahead, Daiichi Sankyo, Sanofi, Merck Sharp & Dome, Tocagen. The following for-profit companies have supported clinical trials and contracted research conducted by MP with payments made to his institution: Boehringer-Ingelheim, Bristol-Myers Squibb, Roche, Daiichi Sankyo, Merck Sharp & Dome, Novocure, GlaxoSmithKline, AbbVie. All other authors declare that they have no conflict of interest related to the present study., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)