The present study was carried out to determine the effect of Acacia nilotica, a tropical plant rich in hydrolyzable tannins (HT), on rumen fermentation and methane (CH4) production in vitro. We used leaves and pods from A. nilotica alone and combined. The combination of HT from A. nilotica leaves and pods and condensed tannins (CT) from Calliandra calothyrsus and Leucaena leucocephala were also evaluated to assess potential differences in biological activity between HT and CT. Four series of 24-h incubations were performed using rumen contents of 4 sheep fed a tropical grass (natural grassland based on Dichanthium spp.). A first experiment tested different levels of replacement of this tropical forage (control [CTL] without tannins) by A. nilotica leaves or pods: 0:100, 25:75, 50:50, 75:25 and 100:0. A second experiment tested the mixture of A. nilotica leaves and pods in different proportions: 100:0, 75:25, 50:50, 25:75, and 0:100. A third experiment tested the 50:50 combination of A. nilotica leaves or pods with C. calothyrsus and L. leucocephala. Acacia nilotica pods and leaves had a high content of HT (350 and 178 g/kg DM, respectively), whereas C. calothyrsus and L. leucocephala had a high content of CT (361 and 180 g/kg DM, respectively). The inclusion of HT from A. nilotica leaves and pods decreased CH4 production dose-dependently (P < 0.01). Total replacement of the CTL by A. nilotica decreased CH4 production by 64 and 55% with leaves and pods, respectively. Pods were richer in HT than leaves, but their antimethanogenic effect did not differ (P > 0.05). Although A. nilotica leaves and pods inhibited fermentation, as indicated by the lower gas production and VFA production (P < 0.01), this effect was less pronounced than for CH4. Volatile fatty acid production decreased by 12% in leaves and by 30% in pods when compared with the CTL alone. Positive associative effect was reported for VFA, when HT-rich sources and CT-rich sources were mixed. Combining the 2 sources of HT did not show associative effects on fermentation or CH4 production (P > 0.05). Hydrolyzable tannin-rich sources were more effective in suppressing methanogenesis than CT-rich sources. Our results show that HT-rich A. nilotica leaves and pods have the potential to reduce ruminal CH4 production.