Human exposure to mixtures of polychlorinated biphenyls (PCBs) may result in the formation of different profiles of hydroxylated PCBs (HO-PCBs), as a consequence of different exposures or dissimilar metabolism of parent compounds. Therefore, we investigated the levels and profiles of PCBs and HO-PCBs in human serum samples collected from two European countries with different degrees of pollution. There was no significant difference between the levels of sum PCBs measured in each set of samples, with a median concentration of 3100 pg/mL for Romanian samples (n = 53) and 3380 pg/mL for Belgian samples (n = 22). However, the median concentrations recorded for sum HO-PCBs were almost double in Belgian (310 pg/mL) compared to Romanian (175 pg/mL) samples. The detection frequency recorded for HO-PCBs in Belgian samples was also significantly higher compared to Romanian samples. The main contributors to the sum HO-PCBs in the Belgian samples were 4HO-CB107 > 4HO-CB146 > 4HO-CB187 (76% from the sum HO-PCBs) and 4HO-CB187 > 4HO-CB146 > 3'HO-CB138 (66% from the sum of HO-PCBs) in the Romanian samples. The HO-PCB profile showed that the higher chlorinated HO-PCBs had a higher contribution in the Romanian samples compared to the Belgian ones. This suggests that differences in the PCB profiles between populations can lead to the formation of different HO-PCB metabolite profiles presenting thus different risks for populations. No clear preferential mechanism of HO-PCB metabolite formation (HO-direct insertion vs. 1,2-shift of a chlorine atom) could be highlighted for investigated samples. The main chlorinated phenolic compound found in the Belgian samples was pentachlorophenol (PCP) which accounted for up to 85% of the total quantified phenolics, whereas in the Romanian samples, PCP accounted for only 35%.