Objective: To study the influence of povidone-iodine (PI) versus that of the benzethonium chloride wipe (BCW) on semen collection and semen quality of sperm donors undergoing penile skin disinfection and provide some evidence for the selection of disinfection methods for semen collection. METHODS: We used PI from August to December 2015 and BCWs from January to July 2016 for penile skin disinfection before semen collection, with two samples from each donor, one collected with and the other without penis skin disinfection (the blank control group). After semen collection, we conducted a questionnaire investigation on the influence of the two disinfection methods on semen collection and compared the semen parameters between the two groups of sperm donors. RESULTS: Totally, 185 sperm donors were included in this study, of whom 63 underwent penile skin disinfection with PI and the other 122 with BCWs before semen collection. Statistically significant differences were found between the PI and BCW groups in the adaptability to the disinfectant and rigid disinfection procedures (P <0.05), but not in the other items of the questionnaire (P >0.05). Compared with the sperm donors of the blank control group, those of the PI group showed statistically significant difference in the percentage of progressively motile sperm (PMS) (ï¼»63.02 ± 3.18ï¼½% vs ï¼»61.45 ± 4.78ï¼½%, P<0.05), but not in the abstinence time (ï¼»4.97 ± 1.79ï¼½ vs ï¼»4.7 ± 0.94ï¼½ d, P >0.05), semen volume (ï¼»4.11 ± 1.54ï¼½ vs ï¼»4.15 ± 1.61ï¼½ ml, P >0.05), sperm concentration (ï¼»110 ± 29.6ï¼½ vs ï¼»107.5 ± 31.79ï¼½ ×106/ml, P >0.05), or total sperm count (ï¼»439.10 ± 170.13ï¼½ vs ï¼»434.02 ± 186.91ï¼½ ×106/ejaculate, P >0.05), while those of the BCW group exhibited no remarkable difference in any of the above parameters (P >0.05). Among the samples with abnormal semen quality, significantly fewer were found with abnormal PMS in the BCW than in the PI group (1.64% ï¼»2/122ï¼½ vs 9.68% ï¼»6/62ï¼½, P <0.05). However, there were no significant differences between the PI and BCW groups in the abnormal semen volume, abnormal sperm concentration, or the rate of semen bacterial contamination (P >0.05). CONCLUSIONS: Before semen collection from donors, penile skin disinfection with povidone-iodine may affect both the semen collection process and the quality of donor sperm, while the benzethonium chloride wipe can reduce the influence on the semen collection process and does not affect the semen parameters.