A risk-oriented evaluation of biofilm and other influencing factors on biological quality of drinking water for dairy cows. Journal of animal science Despite the importance of livestock drinking water quality on animal physiology, welfare, and performance, influences such as biofilm formation on trough surfaces on microbial water quality are rarely researched. The objective of this study was to assess the microbial quality of water offered to lactating dairy cows and identify risk factors for poor water quality. We further aimed to determine the impact of biofilm formation on water quality and evaluate rapid test systems to score the hygiene status of dairy troughs on the farm. A total of 105 troughs located on 24 typical Western German dairy farms were sampled. Samples of livestock drinking water and biofilm were analyzed for aerobic total viable count (TVC), coliform count (CC), Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and other bacteria resistant to 3rd generation cephalosporins (CRB). Surface protein- and adenosine triphosphate (ATP)-rapid tests were evaluated to detect biofilm formation. The influence of 22 selected fixed and variable trough characteristics on impaired livestock drinking water quality was evaluated by calculating odds ratios. The average TVC, CC, and E. coli counts were 4.4 ± 0.06 (mean ± SD), 1.7 ± 0.1, and 0.6 ± 0.1 log10 cfu per mL, respectively. CC was detectable in 94.3% of all water samples and E. coli in 48.6%. MRSA was found in pooled livestock drinking water samples of a single farm and CRB on three farms, suggesting that troughs might function as a reservoir of antibiotic-resistant bacteria, thereby contributing to an exchange of antibiotic-resistant bacteria between animals. Risk factors for the impairment of at least one microbial quality criteria (TVC, CC, or E. coli) increased significantly (P < 0.05) when using high-volume troughs, other trough materials than stainless steel, a lower distance to the milking parlor, heavy visible soiling, biofilm formation, and high ambient and high water temperatures. CC (r = 0.46; P < 0.001) and E. coli (r = 0.31; P < 0.01) of water samples correlated with their equivalent in biofilm and with the results of rapid tests on trough surfaces (0.31 > r > 0.19; P < 0.05). Addressing the identified risk factors could be an approach to ensure sufficient biological quality of livestock drinking water. 10.1093/jas/skac112