The parasitic plant purple witchweed [Striga hermonthica (Delile) Benth.] poses a serious threat to cereal production in sub-Saharan Africa. Under natural infestation, the wild pearl millet [Pennisetum glaucum (L.) R. Br.] line 29Aw demonstrates resistance against the parasite, but the mechanism of its resistance is unknown. Striga resistance can be due to: (1) low induction of Striga germination (pre-attachment resistance) or (2) inhibition of parasite attachment and development (post-attachment resistance). Germination bioassays and root chamber (rhizotron) resistance-screening assays were used to determine the extent of pre- and post-attachment Striga resistance in 29Aw compared with the Striga-susceptible ‘SOSAT-C88-P10’ variety. Regarding pre-attachment resistance, 29Aw stimulated 10-fold less Striga seed germination at a maximum germination distance of 7.96 ± 2.75 mm from the host root compared with 35.94 ± 2.88 mm in SOSAT-C88-P10. Post-attachment resistance revealed 10- to 19-fold fewer and 2.5-fold shorter Striga seedlings with 28-fold less biomass growing on 29Aw compared with SOSAT-C88-P10. Microscopic analysis showed that Striga penetration in 29Aw was blocked at the endodermal and cortical levels. Post-attachment resistance in 29Aw was further supported by fewer (22%) Striga–host vascular connections in 29Aw compared with 79% in SOSAT-C88-P10. Together, these findings demonstrate that 29Aw harbors both pre- and post-attachment resistance mechanisms against S. hermonthica.