BACKGROUND: Atrial fibrillation (AF) is the predominant arrhythmia. Growing evidence indicates that immunological diseases may play a role in its development. This study combined observational and genetic analyses to investigate how T-lymphocyte subsets and immunomodulatory drug targets influenced AF risk. METHODS AND RESULTS: This study retrospectively analyzed T lymphocyte subsets in 302 AF patients and 183 healthy controls. After balancing covariates via 1:1 propensity score matching, multivariate regression revealed AF patients had significantly higher absolute counts of Th1 and Th17 cells, and higher Th1/Th2 and Th17/Treg ratios (p < 0.05). A subsequent two-sample Mendelian randomization (MR) analysis, which assessed 731 immune traits and included drug-target MR, established causal relationships. It showed that increased counts of CD4+CD8dim T cells (OR = 1.03, p = 0.002) and IgD-CD38dim B cells (OR = 1.06, p = 0.0003) elevated AF risk. Furthermore, IL-6R-mediated CRP elevation increased AF risk (OR = 1.33, p = 5.18E-7), whereas higher IL-18 gene expression was protective (OR = 0.95, p = 0.036). CONCLUSION: Immune dysfunction, especially T lymphocyte subsets, is a risk factor for the development of AF. The IL-6R signaling pathway may be a potential target for AF prevention, whereas the use of IL-18 inhibitors may increase the risk of AF.