Tympanosclerosis is a chronic middle ear disorder characterized by fibrosis, hyalinization, and calcification of the tympanic membrane and ossicles, which often results in conductive hearing loss. The objective of this systematic review was to synthesize current evidence on molecular pathways and genetic susceptibility factors contributing to the development of this condition. PubMed, Embase, Web of Science, and Google Scholar were searched up to December 2024. Eligible studies were original peer-reviewed articles in English investigating gene expression, genetic polymorphisms, or molecular signaling pathways in human or animal models. Risk of bias was assessed using standardized tools, and results were synthesized narratively due to heterogeneity. Twenty-five studies were included from 1815 screened records. Reported findings implicated inflammatory cytokines, such as TNF-α and IL-6, oxidative stress-related enzymes, including CAT, and iNOS, and bone remodeling pathways involving Wnt signaling, TGF-β1, and osteopontin. Polymorphisms in TLR4, NOS2 and NAT2 were associated with increased susceptibility or severity. Evidence remains limited but highlights potential biomarkers and therapeutic targets.