Case-control studies previously conducted in Pakistan suggested that topical antimicrobials might provide protection against neonatal tetanus (NNT) when applied to the umbilical cord wound during the first several days of life. The present case-control study, the largest such study yet reported, was undertaken in Punjab Province, Pakistan and afforded further opportunities to evaluate such effects. A population-based, matched, case-control study was undertaken to assess topical antimicrobials and other factors related to NNT risk in rural parts of Punjab Province. Continuous use of antimicrobial agents (antibiotics and antiseptics) at delivery and during the first few days after delivery was highly protective in univariate testing (matched odds ratio 0.2 [95% confidence interval: 0.11-0.64], P = 0.003), and remained significantly protective when other delivery and cord care practices were controlled. In contrast, applying nothing to the wound was risky compared with antimicrobial exposures. Hand washing and delivery by a trained birth attendant appeared protective. Application of animal dung or ash to the umbilical wound was hazardous. Similarly, predelivery cutaneous or intravaginal exposure of mothers to ghee (clarified butter) and delivery on a surface prepared with dried cow dung were risky, with significant interaction noted between them. Mortality and NNT were far more likely among previous births to mothers of cases. Topical antimicrobials offer a new, effective and inexpensive means to prevent NNT, and could usefully complement maternal immunization with tetanus toxoid in controlling the disease. Special prevention efforts should be directed towards mothers of NNT cases.