This retrospective cohort study assessed semen and testicular tissue quality from adult and adolescent cancer patients who had samples cryopreserved in the Cryobank of Charité-Universitätsmedizin before and/or after cancer treatment. Medical and cryopreservation data for all samples stored between 03/2004 and 05/2019 were collected retrospectively. We included information on 601 samples cryopreserved from 506 cancer patients for whom oncologic treatment data were available. The majority of the samples were cryopreserved prior to cancer treatment (460/600, 77%, median 5 days before treatment). Semen quality had a predisposed reduction in those collected from adolescents with testicular and/or hematological malignancies. Analyses of the 140 (23%) samples cryopreserved after treatment initiation (median of 84 days) revealed decreased median concentration and motility following high gonadotoxic-risk treatment. Rate of oligoasthenozoospermia was comparable in samples collected prior to treatment with those provided during follow-up spermiograms within 1 year after treatment initiation (45.5% vs 45.5%). However, an increase was seen in samples collected 1-2 (9.1% to 90.9%) and 2-3 (50.0% to 100.0%) years after treatment initiation. Cancer diagnosis and treatment may impair spermatogenesis; therefore, patient counseling prior to cancer treatment by an oncologist and/or fertility specialist is crucial.