The aim of the study was to investigate the microbial populations of water and biofilm samples from seven hand dug wells in Ilorin, Nigeria, which are for private and public use. Physicochemical parameters such as pH, turbidity, total dissolved solids, temperature, conductivity, dissolved oxygen and biological oxygen demand were determined. Biofilm growths were induced from water samples obtained from selected wells. Twenty bacteria isolated were identified using morphological and biochemical characteristics as well as molecular methods and includes Shigella sonnei, Shigella dysenteriae, Micrococcus luteus, Bacillus sphaericus, Salmonella enteritidis, Proteus mirabilis strain IK-MB4-518F, Bacillus licheniformis strain RH104, Bacillus subtilis, Erwinia sp., Proteus vulgaris, Yersina sp., Bacillus cereus, Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa strain GS1, Pseudomonas aeruginosa strain 218B, Staphylococcus epidermidis, Vibrio sp., Escherichia coli and Bacillus pasteurii. Total bacterial count ranged from 1.8×104-1.53×106 cfu/ml. Water samples from these wells are therefore not potable and some of the isolates are potential health hazards to human population.