The objectives of this study were (1) to evaluate the test characteristics and predictive values of quarter-composite milk somatic cell count (quarter-cSCC) values based on either a single observation or the geometric mean of multiple recordings as a predictor of intramammary infection (IMI) in lactating dairy cows; and (2) to explore to what extent herd prevalence of IMI and cow factors such as parity and stage of lactation affect them. A total of 780 single-quarter milk samples were collected from 195 dairy cows for bacteriologic culture at a single cross-sectional herd screening performed at 21 different dairy herds as part of different research projects. Additionally, monthly quarter-cSCC milk samples at test day were available as part of the Dairy Herd Improvement program. Sensitivity (Se), specificity (Sp), positive predictive valu (PPV), and negative predictive value (NPV) were calculated to differentiate cows infected with any pathogen and cows infected with major pathogens from uninfected cows. Different threshold values for quarter-cSCC, ranging between 50,000 and 500,000 cells/mL, were evaluated for all animals in the study, as well as for high- and low-prevalence herds, heifers and multiparous cows, and cows in early, mid, and late lactation. The overall Se and Sp at a threshold of 200,000 cells/mL for a single quarter-cSCC observation obtained closest to the time of bacteriologic culture were 44.3 and 87.3%, respectively, for cows infected with any pathogen, and 65.1 and 73.0%, respectively, for cows infected with major pathogens. The overall PPV and NPV at a threshold of 200,000 cells/mL for a single quarter-cSCC observation obtained closest to the time of bacteriologic culture were 89.9 and 38.1%, respectively, for cows infected with any pathogen, and 40.6% and 88.1%, respectively, for cows infected with major pathogens. No major differences were observed between estimates of the test characteristics and predictive values of the quarter-cSCC criteria based on a single observation and the geometric mean of multiple observations. For IMI with any pathogen, the Se and PPV were higher in high-prevalence herds than in low-prevalence herds, particularly at thresholds of 50,000 and 100,000 cells/mL. For IMI with major pathogens, Sp was substantially higher in low-prevalence herds than in high-prevalence herds. Sensitivity was higher in multiparous cows than in heifers infected with any pathogen, more specifically at a threshold of 100,000 and 200,000 cells/mL. For cows in early and mid lactation infected with any pathogen, Sp was higher than for cows in late lactation using the single observation closest to the time of bacteriologic culture. The results suggest that the quarter-cSCC threshold value to select cows for bacteriologic culture to maximize the likelihood of finding the causative pathogen of IMI should depend on the group of pathogens one is interested in, the herd prevalence of subclinical mastitis, lactation stage, and the cow's parity.