This study had the objective to evaluate the effect of Piper betle L. powder (PP) at 5 different doses in substrate incubated by sunflower oil as secondary function of PUFA using in vitro gas production technique. The treatments of this study were run as a 2X5 factorial arrangement in a completely randomised design using the PROC GLM procedure of SAS 9.4: (1) control (S1) without supplementation of PP; (2) 15 mg PP (S2); (3) 30 mg PP (S3); (4) 45 mg PP (S4); and (5) 60 mg PP (S5), while sunflower oil was supplemented in all treatments: low 15 mg/incubation and high 30 mg/incubation. A 500 mg of TMR (hay: concentrate, 50:50) was assigned to basal substrate. The PP containing 1.84 mg/g DM quercetin and 1.00 mg/g DM eugenol altered rumen fermentation without change pH (p < 0.001) and methane production was lesser (p < 0.001) about -30% and -25% for DM and OM measurement, respectively. Gas kinetic, degradability, and ammonia level was significantly affected by supplementing PP (p < 0.01). Overall, this study suggested quercetin and eugenol deriving from PP acted three major accelerations: assembled carbon dioxide, behaved antimicrobial role and performed the balance water molecules in the rumen kinetic. This study suggests that PP promotes changing in vitro rumen fermentation and diminishing methane production within recommended doses, 0.1-15 mg/incubation in DM.