The present investigation focused on groundwater hydro-geochemistry of Alsisar block of Jhunjhunu district, India, aims on evaluating the quality of groundwater for drinking and irrigation purposes and assessing the human health risk from ingestion of groundwater. The groundwater of Alsisar block is neutral to alkaline, brackish and very hard in nature. Total dissolved solids, total hardness, Na+, Mg2+, HCO3−, F− and NO3− in majority of the groundwater samples were exceeding the World Health Organization and Bureau of Indian Standards recommended limits. The drinking water quality index ranged from 111.53 to 492.84. None of the sample belonged to excellent and good categories of drinking water quality. Fluoride varied from 0.018 to 4.176 mg L−1, and nitrate varied from 0.34 to 520.66 mg L−1 in groundwater. The non-carcinogenic risk assessment for children, men and women owing to ingestion of fluoride and nitrate-enriched groundwater indicates human health risks in the entire study area. Irrigation with groundwater of Alsisar block is liable to cause salinity and magnesium hazard to agricultural crops grown in the area. Source apportionment using principal component analysis suggests the geogenic origin of fluoride and anthropogenic origin of nitrate. Na+–Mg2+–Cl− followed by Na+–Mg2+–HCO3− are the predominant hydrochemical facies in the groundwater of Alsisar block. Silicate rock weathering, ion exchange and evaporation are the predominating processes governing ionic concentrations in the groundwater. Biochemical and molecular tests demonstrated the presence of Brevibacillus borstelensis strain DSM 6347 16s rRNA and Bacillus paramycoides strain MCCC 1A04098 16s rRNA in the groundwater of the area.