Allelopathic effects of Cyperus rotundus on banana ‘Grande Naine’ were studied in vitro as well as ex vitro. To study the allelopathic effects in vitro, the Cyperus extract was added to the multiplication medium during preparation before adjusting the pH. Of the four concentrations, 0.2 and 0.6% decreased the shoot multiplication and shoot length but 1 and 2% extract completely inhibited the shoot multiplication but induced rooting in 50 and 28% shoots, respectively. For the ex vitro studies, the Cyperus extract was added to the hydroponic medium during the hardening of in vitro raised banana plantlets. The extract of 1 and 2% concentrations decreased the shoot and root length, number of leaves and new roots, fresh weight, total chlorophyll and protein. The fluorescence intensity ratio was successively increased resulting in decreased photosynthesis.