Background: Testicular microlithiasis is the presence of small calcifications in the testicular parenchyma. The association between testicular microlithiasis and germ cell neoplasia in situ, a precursor to testicular cancer, is still unclear. Objectives: To determine the incidence of germ cell neoplasia in situ in men with testicular microlithiasis and evaluate the indication for testicular biopsy according to risk factors in the form of male infertility/reduced semen quality, testicular atrophy, and history of cryptorchidism. Materials and methods: This retrospective case series included all patients diagnosed with testicular microlithiasis who underwent testicular biopsies at three hospitals in Denmark between 2007 and 2021. The medical records of 167 patients were reviewed, and data on patient demographics, testicular microlithiasis characteristics, risk factors, histological findings, and treatments were collected. The main outcome measure was the incidence of germ cell neoplasia in situ in relation to each risk factor. The data were analyzed using descriptive statistics. Logistic regression was used to examine the odds ratio of germ cell neoplasia in situ in patients with testicular microlithiasis and testicular atrophy. Results: Germ cell neoplasia in situ was found in 13 out of 167 patients (7.8% [95% confidence interval: 4.3, 13.2]). Eleven of these had testicular atrophy resulting in a significantly higher incidence in this group than other risk factors (odds ratio 9.36 [95% confidence interval: 2.41, 61.88]; p = 0.004). Discussion: The study comprises the largest cohort to date of men who have undergone testicular biopsies because of testicular microlithiasis and additional risk factors. Limitations include its retrospective design, and relatively low absolute numbers of patients with germ cell neoplasia in situ on biopsies. Conclusion: This study found that men with testicular microlithiasis and testicular atrophy are at an increased risk of germ cell neoplasia in situ. Additionally, our results indicate that biopsies should be considered in men with a combination of subfertility and bilateral testicular microlithiasis. Our findings do not support testicular biopsies for men with testicular microlithiasis and other risk factors.