Guava (Psidium guajava) wilt, caused by Nalanthamala psidii, has been a destructive disease in Taiwan, Thailand, Malaysia and South Africa since it was first reported in guava in 1926. However, the primary infection site of the pathogen remains controversial. This study was conducted to elucidate the importance of root infection by N. psidii in guava orchards in Taiwan. During 2010–2012, a total of 23 guava trees were sampled from three orchards and the isolation frequency of N. psidii from wounded twigs and roots was analysed. The overall isolation frequency of N. psidii was significantly higher (P < 0·01) from roots than trimmed and pruned twigs. Nalanthamala psidii was detected in 53·5% of the root samples from a guava tree showing mild symptoms of wilt and in 0·9% of the root samples from another guava tree without visible symptoms of wilt; however, no N. psidii was detected in the tissues of the trimming and pruning wounds in either tree. Cross sections of diseased guava branches showed that N. psidii colonized the lower part more heavily than the upper part of the branch. When guava trees were inoculated by applying N. psidii-infested soil to injured roots, wilt symptoms developed within 6–13 months. Furthermore, N. psidii infection of guava seedlings via root contact with wilted guava trunks was recorded for the first time in Taiwan, demonstrating the importance of root infection in guava wilt disease.