Field study was carried out to determine the long term effect of herbicide application on soil microbial community in rice- rice cropping sequence in acid soil of North-East India. Treatment comprised of absolute control with one hand weeding, butachlor + 2,4-D with 100% NPK through chemical fertilizer, butachlor + 2,4-D with 75% NPK through chemical fertilizer and 25% through organic source, butachlor + 2,4-D rotated with pretilachlor with 100% NPK through chemical fertilizer and butachlor + 2,4-D rotated with pretilachlor with 75% NPK through chemical fertilizer 25% through organic source. Result revealed that, after 14 years of continuous use of herbicide and organic input along with recommended dose of fertilizer application demonstrated significant increase in the activity of acid phosphatase. The effect of herbicide application was more prominent with sole chemical fertilizer than with organic manure for 25% N fertilizer replacement. Dehydrogenase activity in soil was increased following herbicide application up to 14 days after that it again decreased more prominent with addition of organic manure than with chemical fertilizer. No characteristic trend of urease activity was observed after application of herbicide. Significant inhibition of respiration was observed after application of herbicide up to 14 days followed by gradual recovery afterwards. Microbial biomass carbon in soil was significantly enhanced by application of organic manure for 25% N fertilizer substitution. Under rice-rice cropping system, application of herbicide showed temporary decline in microbial population and enzyme activities up to 14 days. Application of organic manure for 25% N fertilizer substitution significantly enhanced the microbial population and enzyme activities as compared to sole application of chemical fertilizers. Further application of butachlor rotated with pretilachlor resulted in higher microbial population as well as enzyme activities.