The agricultural sector is one of the sources of greenhouse gases (GHGs) emission, especially methane (CH4), and contributing approximately 250 million metric ton carbon dioxide (CO2) equivalent emission per year. Almost 70% of CH4 emission from this sector is enteric fermentation, while 26% is from the livestock manure management. Both rumen and animal manure are the impending sources of carbon (C), sulfur (S), and water (H2O) and microbial populations utilize these constituents to produce GHGs, and hydrogen sulfide (H2S). Nanoparticles (NPs) application in manure is a promising treatment option for mitigating GHG and H2S gases, but limited information is available on how the reduction mechanism occurs. In this study, zinc silica nanogel (ZnSNL), copper silica nanogel (CuSNL), and nano acetyl cysteine (NACL) coated zinc oxide quantum dots (Qdots), zinc oxide (nZnO), and silver (nAg) NPs were tested in manure stored under anaerobic conditions to understand the reduction mechanism of GHG and H2S resulting from NPs application. Additionally, in vitro study with nZnO and two types of feed (alfalfa and corn silage) were conducted to investigate the efficacy of nZnO in mitigating ruminal gas emission. Methane and CO2 concentrations were measured using an SRI-8610 gas chromatograph and H2S was measured using a Jerome 631X meter. Microbial populations were characterized using both plate counts and quantitative real-time polymerase chain reaction (qRT-PCR). Application of NPs reduced gas volumes ranging 16 to 99%, and concentrations reduced by 49 to ~100% for H2S, and 20.24 to ~100% for GHGs. Application of NPs reduced 38.49 to 94.32% aerobic- and 7.43% to 82.04% anaerobic-microbial populations. Furthermore, the qRT-PCR analysis showed that reduction of gases was due to the inhibition of gas specific microbial population. Overall, nZnO based treatments reduced 8.80 to 55.64% methyl coenzyme M reductase (mcrA) gene copies and 0.74 to 25.16% dissimilatory sulfide reductase (DSR). Contrariwise, compared to the control treatment, in vitro study demonstrated 4.89 to 53.65% H2S and GHGs concentration reduction with the applied nZnO inclusion rates. Additionally, alfalfa as feed exhibited 37 to 45% cumulative gas reduction than corn silage but increased GHGs generation 2.17 to 23.17% and ~60% H2S concentration.