Microsurgery of andrology always brings unexpected findings. Scrotal calculi are rare and unique, which are easily confused with tumor. To understand its etiology and harm, our study retrospectively analyzed the clinical characteristics of men with scrotal calculi to provide a reference for clinical practice. Methods: The clinical data of patients who underwent microscopic testicular sperm extraction (MTESE) and microscopic epididymal sperm aspiration (MESA) from January 1, 2018 to December 31, 2021 were retrospectively analyzed. Data screening was performed on cases in which calculi were found or not, and the relationship between calculi and spermatogenesis was analyzed. Results: A total of 405 patients were recruited. After screening, 218 nonobstructive azoospermia (NOA), 83 obstructive azoospermia (OA), and 13 cryptozoospermia (CZ) patients were included in the study. Calculi were found in 3 patients [incidence was 0.74% (3/ 405)], in which 2 patients had obstructive azoospermia (1 was epididymal calculi, 1 was intrascrotal calculi) and 1 patient had cryptozoospermia (intrascrotal calculi). Pathological results showed that chronic granuloma with abscess infiltration appeared in epididymal tissue, basement membrane thickening and fibrosis appeared in seminiferous tubules, and fibrous hyperplasia with calcium deposition was found in scrotal calculus. White blood cells, lymphocytes, red blood cells, abstinence time and urethritis were closely related to the occurrence of calculi. While abstinence time might be a potential predictor, which increased the risk by approximately 1.2 times. Conclusion: Disturbance of the testicular microenvironment caused by lymphocyte infiltration may be the main reason for scrotal calculi and ultimately cause spermatogenesis disorders. Prolonged sexual abstinence was a potential risk.