Human biomonitoring of environmental exposure remains challenging due to the complex, multisystem biological effects of pollution. Within the exposomic framework, volatilomics, the comprehensive analysis of volatile organic compounds (VOCs) in biological matrices offers a sensitive and minimally invasive approach to investigate exposure-related metabolic patterns and biological responses. This study, conducted within the EXPOMAP study, analyzed biosamples from adults of both sexes residing in two Southern Italian areas characterized by contrasting environmental pressures: the highly impacted Land of Fires (LF) and the less affected Sele river Valley (VLS). VOC profiles in urine (both sexes, n = 65) and semen (male, n = 30) were obtained by HS-SPME-GC–MS and compared across the two populations. Both shared and matrix-specific VOCs were identified, suggesting biological specificity in the responses captured by the two biofluids. Multivariate statistical analysis (PLS-DA) revealed distinct volatilomic patterns in both urine and semen. The urinary dataset suggested a separation trend between LF and VLS groups (R 2 Y ≈ 0.83, Q 2 ≈ 0.56) although the permutation test did not reach the conventional threshold for statistical significance, whereas the seminal dataset showed a clearer discrimination within the present dataset (R 2 Y ≈ 0.97, Q 2 ≈ 0.63). Linear Discriminant Analysis (LDA) confirmed the classification results obtained by PLS-DA showing comparable accuracy, although potential overfitting cannot be excluded due to the limited sample size. The results demonstrated the feasibility of minimally invasive, parallel multi-matrix volatilomics in human biomonitoring. The comparison between urine and semen indicated that these biofluids capture complementary information, suggesting that the use of multiple biofluids could provide a more comprehensive view of exposure-related patterns within the human exposome.