ABSTRACT The excessive proliferation of Pontederia crassipes poses serious threats to freshwater bodies in many parts of the world. This aquatic weed causes severe ecological and economic losses worldwide. In South Africa, research has focused on the release of insect biological control agents associated with P. crassipes, with eight insect agents, and only one fungus released to date. Fungi have been explored as a safe method for the management of P. crassipes, due to their ability to cause and spread a vast array of weed diseases. In this study, naturally occurring fungi associated with P. crassipes were surveyed, isolated, and identified using culture methods to determine their prevalence and pathogenicity to P. crassipes. Forty-two fungal isolates were morphologically identified as belonging to genera Alternaria, Fusarium, Epicoccum and Colletotrichum. Subsequently, molecular techniques and bioinformatics confirmed the identity of the fungi that were most pathogenic as Fusarium incarnatum, Fusarium oxysporum and Fusarium equiseti. In whole plant bioassays, these fungi caused 68, 63 and 54% disease infection, respectively, by the end of six weeks, and thus may be considered for future developments into bioherbicides. Moreover, F. incarnatum exhibited 97% disease severity during in vitro assays. This study is also the first to report F. incarnatum occurrence on P. crassipes in South Africa.