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Prototheca spp. are algae that cause incurable acute or chronic mastitis in dairy cows. The aim of this case-control study was the identification of cow- and herd-level risk factors for this unusual mastitis pathogen. Aseptically collected composite milk samples from 2,428 milking cows in 23 case and 23 control herds were collected between January and May 2011. A questionnaire was administered to the producers, and cow-level production and demographic data were gathered. In 58 of 64 isolates, Prototheca spp. and Prototheca zopfii genotypes were differentiated using PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. All isolates were identified as Prototheca zopfii genotype 2. The mean within-herd prevalence for Prototheca spp. was 5.1% (range 0.0-12.5%). Case herds had a significantly lower herd-level prevalence of Staphylococcus aureus and a higher prevalence of yeasts than did control herds. The final logistic regression model for herd-level risk factors included use of intramammary injections of a non-intramammary drug [odds ratio (OR) = 136.8], the number of different injectable antibiotic products being used (OR = 2.82), the use of any dry cow teat sealant (external OR = 80.0; internal OR = 34.2), and having treated 3 or more displaced abomasums in the last 12 mo OR = 44.7). The final logistic regression model for cow-level risk factors included second or greater lactation (OR = 4.40) and the logarithm of the lactation-average somatic cell count (OR = 2.99). Unsanitary or repeated intramammary infusions, antibiotic treatment, and off-label use of injectable drugs in the udder might promote Prototheca udder infection.