Background: Gas station workers are exposed to carcinogenic substances with impact on the hematologic and immune systems. The aim was to apply the immunophenotyping as a tool in the biological monitoring. Methods: This is a workplace-based case-control study with 49 workers and 26 controls. Medical interviews, hematological exams, and immunophenotyping analyses were performed. According to risk behavior (cleaning flannel and mistrust in the automatic fuel supply) the workers were divided into two groups: low risk (group 1) and high risk (group 2). Results: The results showed that CD16, HLA-DR, CD25, CD56+, CD16 CD56 low, and CD56 high expressions were higher in workers when compared to the control group (P =0.020, P =0.001, P =0.001; P =0.034, P=0.023, and P =0.008, respectively). The expressions of CD2, CD8, CD10, CD8low, and CD4/CD8 ratios were lower (P =0.016, P =0.001, P=0.001, P= 0.017, P = 0.0259, and P =0.029, respectively). Headache and paresthesia complaints were associated with workers when compared to the control group (OR = 4.091, 95% CI, 1.400 -11.951, P = 0.014; OR =12.12, 95% CI, 1.505 - 97.61, P =0.004). Using cleaning flannel and mistrust in the automatic fuel supply (risk behaviors) were associated with group 2 (OR = 9.71, 95% CI, 2.60-36.26, P = 0.005; OR = 18.18, 95% CI, 2.04-161.37, P = 0.004). Conclusions: The results strengthen the worker’s immunosuppression hypothesis, which may contribute to some disorders and the carcinogenesis process. The evaluation of the immune system by flow cytometry is a promising tool for monitoring blood malignancy risk in addition to regular classic hematological exams.