Environmental problems many times could evolve when manure-containing pathogens are distributed into an open environment with no effort made to reduce the content of pathogens or limit their movement in the environment. Wind, surface flow, and subsurface flow can all carry enough pathogens to receiving waters to exceed water quality standards. This study was conducted to assess the microbiological quality of water associated with animal-based agriculture in the sub-basin of Pinhal River located in the rural area of Concordia, Santa Catarina, Brazil. Six sampling points representing different agricultural land uses (LU1-dairy cattle; LU2-without animals; LU3-dairy + pigs + poultry + crops; LU4-pigs + poultry + crops; LU5-dairy + pigs + poultry + crops + human; and LU6-dairy+pigs+crops) along the Pinhal River sub-basin (north to south) were sampled biweekly from August 2006 to December 2008. Concentrations of fecal coliforms and Escherichia coli varied significantly (p≤0.05) with land use (LU), but there was no interaction effect of LU, season, and time. Water samples from the catchment area of LU1 had the highest concentration of fecal coliforms (4,479±597 CFU ml −1 ) when compared with other catchment areas. Catchment area associated with LU2 (no animal) had the lowest concentrations of fecal coliforms (39.2±5.2 CFU ml −1 ). With the exception of LU2 (control site), all the maximum concentrations of E. coli exceeded the single maximum allowable concentration for E. coli (100 CFU ml −1 ). When LU1 was compared with other catchment areas (LU3, 50%; LU4, 67%; LU5, 58%; and LU6, 44%), it had the lowest counts (39%) of Salmonella sp. Our results suggest that spatial pattern of bacterial water quality is evident, which can be linked to the different land uses and associated practices (present or absent of animal activities). Therefore, varying responses associated with the different land uses would be critical in identifying the importance of different sources of bacteria in the catchment area and the mechanisms transferring them.