Within a One Health perspective, stray dogs provide evidence of shared environmental contamination and emerge as sentinels of pollution-related infertility and carcinogenesis in exposed populations. Our work aimed to evaluate correlations between environmental pollutants, such as heavy metals (HMs), and testicular diseases in dogs. To this end, fifty male stray dogs from highly polluted areas in the Campania Region were enrolled. Testicular samples (n = 100) were processed for histological, cytological, molecular, and chemical analyses. Histopathological lesions were graded according to the Johnsen scoring system, using an EfficientNet-B4 deep learning model for automated assessment. Expression levels of 17-β-HSD and P450 aromatase were evaluated by immunohistochemistry (IHC) and Western blot (WB). Sperm chromatin was assessed by toluidine blue staining using an unsupervised k-means clustering algorithm to automatically classify chromatin status and reduce operator subjectivity. Finally, inductively coupled plasma mass spectrometry (ICP-MS) was carried out to assess HM concentrations in testes. Based on histological examination, samples were divided into three groups: A) normal testes (37%); B) testes with moderate to severe degeneration (54%), and C) testicular neoplasm (9%). Groups B and C showed significant downregulation of 17-β-HSD and upregulation of P450 aromatase compared with Group A, alongside reduced sperm chromatin condensation. ICP-MS results showed higher levels of arsenic, cadmium, lead, mercury, and tin in Group C (100%) compared with Group B (67%) and Group A (27%). Notably, all neoplastic samples displayed aberrant uranium accumulation. Our findings suggest a possible correlation between higher HM accumulation and testicular diseases in dogs from polluted areas in the Campania Region, corroborating current scientific literature indicating that HMs may have negative effects on spermatogenesis. This highlights the important role of dogs as sentinels for human reproductive health, underscoring the need for One Health surveillance to assess the silent threat of HM toxicity in the exposed human population.