<p>The study investigates the complex microbial community dynamics and metabolic interactions crucial for efficient biomethane production from water hyacinth. Different inoculum-substrate ratios were analyzed using cow dung as inoculum to optimize biomethane production from water hyacinth. Maximum biochemical methane potential (370 L/kg VS) and microbial diversity are significantly increased at the ideal inoculum-substrate ratio (3:1). Metagenomic analysis reveals dominant bacterial genera like <i>Enterococcus</i>, <i>Comamonas</i>, and methanogens such as <i>Methanobrevibacter</i> and <i>Methanosarcina</i> which play an essential role in bio-methane production. Volatile fatty acid accumulation was also maximum for the inoculum-substrate ratio of 3:1 after the 30th day of incubation (3820&#xa0;mg L⁻¹), predominantly acetic and propionic acids. Metagenome data unveil the presence of cellulolytic microbes and acetogenic bacteria like <i>Ruminococcus</i>, <i>Sporomusa</i>, and <i>Acetobacterium</i>. Understanding these interactions facilitates the enhancement of anaerobic digestion procedures and the establishment of microbial consortiums to generate sustainable biomethane. Furthermore, with an optimal inoculum-substrate ratio of 3:1, up to 3.70 kWh of power can be generated, offering a promising alternative to conventional energy sources to fulfill the sustainable development goal (SDG 7).</p> Graphical abstract <p></p>