The many interesting properties of chitosan polysaccharides have prompted their extensive use as biomaterial building blocks, for instance as antimicrobial coatings, tissue engineering scaffolds, and drug delivery vehicles. The translation of these chitosan-based systems to the clinic still requires a deeper understanding of their safety profiles. For instance, the widespread claim that chitosans are spermicidal is supported by little to no data. Herein, we thoroughly investigate whether chitosan oligomer (CO) molecules can impact the functional and structural features of human spermatozoa. By using a large number of primary sperm cell samples and by isolating the effect of chitosan from the effect of sperm dissolution buffer, we provide the first realistic and complete picture of the effect of chitosans on sperms. We found that CO binds to cell surfaces or/and is internalized by cells and affected the average path velocity of the spermatozoa, in a dose-dependent manner. However, CO did not affect the progressive motility, motility, or sperm morphology, nor did it cause loss of plasma membrane integrity, reactive oxygen species production, or DNA damage. A decrease in spermatozoa adenosine triphosphate levels, which was especially significant at higher CO concentrations, points to possible interference of CO with mitochondrial functions or the glycolysis processes. With this first complete and in-depth look at the spermicidal activities of chitosans, we complement the complex picture of the safety profile of chitosans and inform on further use of chitosans in biomedical applications.