Three nitrate degrading bacteria, namely S1, S2 and S3 strains, were isolated from soil samples collected from agricultural sites at Polonnaruwa, Oruwala and Gampaha, Sri Lanka respectively. Among the isolated strains, S1 showed a maximum nitrate removal rate of 4.20±0.08 mg/L/day whereas S2 and S3 showed nitrate removal rates of 3.45±0.57 mg/L/day and 3.72±0.19 mg/L/day, respectively. The nitrate removal abilities of all three strains were measured at different temperatures (25 o C, 30 o C, 32 o C), different pH (7.0, 7.5, 8.0) and for different nitrate concentrations (15, 30, 45 mg/L) in vitro. The maximum nitrate degrading rate by the bacterium S1 (4.52±0.01 mg/L/day), S2 (4.25±0.47 mg/L/day) and S3 (4.04±0.09 mg/L/day) were detected at 32ºC whereas the highest degradation rate for S1 (4.6±0.05 mg/L/day), S2 (4.38±0.03 mg/L/day) and S3 (4.14±0.25 mg/L/day) bacteria strains was reported when the pH was 8.0. The maximum nitrate degradation was found to be 5.88±0.17, 5.67±0.45 and 5.71±0.43 mg/L/day for S1, S2 and S3 bacteria strains when 45 mg/L of nitrate was present in the medium. A biochemical test tentatively identified the isolates S1, S2 and S3 as Pseudomonas sp., Bacillus sp. and Proteus sp. The bio sand filter developed in cooperating all three bacterial strains showed 6.12±0.06 mg/L/day of nitrate degradation rate within 24 hours.