Background: The present study investigated the relationships between psychological stress indices and oxidative stress marker, also when combined with emergent insulin resistance (IR), in the non-diabetic, middle-aged subjects, exposed to frequent/chronic psychological stressors. Methods: Cross-sectional data from a cohort of non-diabetic police officers (n = 234; 19F), aged 27-56 years, were used. Plasma inflammatory (CRP, TNF-α), oxidative stress (free 8-iso-prostaglandin F 2α ; 8-iso-PGF 2α) markers, and insulin were measured. The value of homeostasis model assessment of IR index (HOMA-IR) was assumed the threshold value of IR, i.e. 2.04. Free cortisol in urine and perceived stress (psychological stress indices) were also measured. Results: In the IR subjects, most biochemical variables, inflammatory markers and urine cortisol were significantly higher, as compared to the non-IR ones. Psychological stress indices were associated with plasma 8-iso-PGF 2α [B = 0.139, 95% CI (0.048, 0.230), p = 0.002, and B = 0.007, 95% CI (0.0006, 0.014), p = 0.03; for perceived stress level and cortisol, respectively]. Positive associations were established between plasma 8-iso-PGF 2α [B = 0.069, 95% CI (0.016-0.120), p = 0.01] and urine cortisol [B = 0.003, 95% CI (0.0003, 0.005), p = 0.02] with HOMA-IR. Metabolic syndrome, as defined by IDF criteria, was established in 110 study subjects, whereas 136 of them were hypertensive. Waist circumference [B = 0.056, 95% CI (0.039, 0.074), p < 0.0001], and systolic blood pressure [B = 0.009, 95% CI (0.00003, 0.018), p = 0.04] were positively associated with HOMA-IR, whereas the association of HDL cholesterol [B = − 0.597, 95% CI (− 1.139, − 0.055), p = 0.03] was a negative one. Cortisol [OR = 1.007, 95% CI (1.002, 1.012), p = 0.006], and 8-iso-PGF 2α [OR = 1.103, 95% CI (1.010, 1.201), p = 0.02] affected the incidence of IR. After adjustment for metabolic syndrome (or its components), age, sex, and current smoking, the effects became non-significant. Out of metabolic syndrome components, waist circumference [OR 4.966, 95% CI (2.29, 10.751), p = 0.00004] and hypertriglyceridemia [OR 1.993, 95% CI (1.063, 3.736), p = 0.03] increased the chance of IR incidence. Conclusions: Both psychological stress indices were associated with oxidative stress, but only cortisol with HOMA-IR. In the subjects exposed to frequent/chronic psychological stressors, cortisol and oxidative stress marker affected IR incidence, being statistically attenuated, though, following adjustment for metabolic syndrome, or its components.