Benzo(a)pyrene (BaP) is considered one of the most dangerous air pollutants for adverse health effects, including reproductive toxicity. It is found both in male and female reproductive fluids likely affecting spermatozoa after the selection process through cervical mucus, a process mimicked in vitro with the swim-up procedure. In vitro effects of BaP (1, 5, 10 µM) were evaluated both in unselected and swim-up selected spermatozoa after 3 and 24 h of incubation. BaP reduced total, progressive and hyperactivated motility and migration in a viscous medium both in swim-up selected and unselected spermatozoa. Viability was not significantly affected in swim-up selected but was reduced in unselected spermatozoa. In swim-up selected spermatozoa, increases in the percentage of spontaneous acrosome reaction and DNA fragmentation were observed after 24 h of incubation, whereas no differences between the control and BaP-treated samples were observed in caspase-3 and -7 activity, indicating no effects on apoptotic pathways. ROS species, evaluated by staining with CellROX® Orange and Dihydroethidium, did not differ in viable spermatozoa after BaP treatment. Conversely, the percentage of unviable ROS-positive spermatozoa increased. Our study suggests that BaP present in male and female genital fluids may heavily affect reproductive functions of human spermatozoa.