Male infertility is a global issue that seriously affects reproductive health. This study aimed to understand the underlying causes of idiopathic non-obstructive azoospermia (iNOA), which is a type of male infertility with unknown origins that accounts for 10–15% of cases. By using single-cell analysis techniques, we aimed to uncover the mechanisms of iNOA and gain insight into the cellular and molecular changes in the testicular environment. In this study, we performed bioinformatics analysis using scRNA-seq and microarray data obtained from the GEO database. The analysis included techniques such as pseudotime analysis, cell–cell communication, and hdWGCNA. Our study showed a significant difference between the iNOA and the normal groups, indicating a disorder in the spermatogenic microenvironment in iNOA. We observed a reduction in the proportion of Sertoli cells and blocked germ cell differentiation. Additionally, we found evidence of testicular inflammation related to macrophages and identified ODF2 and CABYR as potential biomarkers for iNOA.