Background: Spermicides have been identified as a potentially attractive alternative to hormonal contraceptives and/or intrauterine devices. Thus, this study aimed evaluating the efficacy and local tolerance of benzalkonium chloride (BKC) and myristalkonium chloride (MKC) contained in Pharmatex V R vaginal formulations and compare them with nonoxynol-9 (N-9), the most common active ingredient in topical vaginal contraceptives. Methods: Human normozoospermic samples were assessed for motility, viability, acrosome status and penetration ability after exposure to control, N-9 or different BKC and MKC doses for 0 and 10 minutes. Local tolerance on HeLa cells was evaluated by the Trypan-blue and MTT assays. Results: Exposure to BKC and MKC reduced acrosome integrity while promoting total immobilisation and complete loss of sperm viability (p < .001, n ¼ 15). Both compounds also compromised sperm penetration ability upon exposure (p < .001, n ¼ 15). N-9 induced the same outcomes (p < .001, n ¼ 15); nevertheless, it was more toxic to HeLa cells than BKC and MKC (p < .05, n ¼ 14). Conclusions: BKC and MKC present strong in vitro spermicidal activity at lower doses than N-9 and were better tolerated after immediate exposure than N-9. Available Pharmatex V R galenic formulations were as effective as products based on N-9.