Background: Chemical castration via in-situ non-invasive methods has been proposed as a viable alternative to surgical castration, owing to its reduced post-operative complications and lower associated costs. This study was designed to investigate the effect of intra-testicular injection of carbon quantum dots (CQDs)—synthesized from white onion (Allium cepa) juice—on the spermatological parameters of mature New Zealand White (NZW) rabbits. Methods: In this experiment, which extended over a ۵۰-day period, eight mature male NZW rabbits of comparable age and weight were randomly allocated to one of three experimental groups: the untreated control (without undergoing any experimental procedure); the sham group (which received needle insertion without any solution to control for the procedure’s mechanical impact); and the CQDs-treated group (which received carbon quantum dots, synthesized via a one-step hydrothermal carbonization method, at a dose of ۴۰.۰۰ mg/kg per testicle). A comprehensive spermatological evaluation was performed, encompassing the analysis of several parameters: various motility characteristics (including total and progressive motility, as well as kinematic patterns), in addition to assessments of functional and structural integrity—namely, plasma membrane functionality, DNA integrity, cell viability and morphological normality. Results: According to the findings, the intra-testicular injection of carbon quantum dots (CQDs) exerted a significant impact on sperm, which included a pronounced reduction in kinematic characteristics, plasma membrane functionality, and viability. Concurrently, this injection was associated with a marked increase in levels of DNA damage and morphological abnormalities. Conclusion: This research indicates that CQDs are promising candidates as chemical castration agents in mature male NZW rabbits. However, further studies are needed to evaluate their long-term efficacy and investigate any potential systemic side effects.