The structural, morphological, thermal, and anti-microbial properties of NiO nanoparticles were investigated using natural fuels in conjunction with the combustion process. XRD revealed the Face-centered cubic polycrystalline structure in all of the samples. The crystallite diameters for tea extract, wine, honey, and cow urine-assisted nanoparticles were 32.60 nm, 27.41 nm, 29.58 nm, and 27.75 nm, respectively, according to the WH plot for NiO nanoparticles. The FESEM and EDAX analytical results corroborate the stoichiometric of the products as well as the existence of diamond, pollen grain, cluster pore, and mushroom-like morphologies. The average particle size as assessed by TEM and the size of crystallites are connected. All XRD pattern parameters that were matched with JCPDS data (04–0835), as well as the FTIR spectrum, support the presence of a pure phase of Face-centered cubic NiO in the range of 453.57 cm-1 to 438 cm-1. Thermal investigations (TG-DTA) indicated that the particles were stable even at 1000 °C. When NiO is subjected to thermal analysis, there is evidence of weight gain and loss. Anti-microbial study of organically fueled NiO used Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) bacteria, as well as Candida albicans and Aspergillus niger fungi. It has been discovered that NPs are more efficient at inhibiting fungal growth than bacterial development.