The aim of this study was to investigate the variation of gross composition, somatic cell count, urea content, and fatty acids (FA) composition of bulk milk from single-breed and multi-breed farms in alpine area, keeping either Brown Swiss (BS), Holstein Friesian (HF), Simmental (SI), or their combinations. Gross milk composition, urea content, and FA composition were predicted using mid-infrared spectroscopy. Observations were grouped in 7 combinations consisting of 3 single-breed and 4 multi-breed types of herd. A mixed linear model was used for data analysis, accounting for the fixed effects of herd composition (7 combinations), month of sampling, year of sampling, and the interactions between herd composition and month of sampling, and between herd composition and year of sampling. Farm was included as random effect. Results highlighted that about two thirds of South Tyrolean farms were single-breed and herds with more than 20 lactating cows changed herd structure over time, switching from multi- to single-breed. Single-breed BS farms produced milk with greater fat, protein, casein, lactose, and FA content than single-breed HF and SI farms. Further, multi-breed herds including BS cows produced milk with greater fat, protein, casein, and polyunsaturated FA content than multi-breed HF+SI herds. Overall, single-breed SI farms produced milk with lower somatic cell count than other herd combinations. Despite the number of BS cows in South Tyrol has decreased in favor of SI in the last years, this breed is still the most interesting for alpine dairy farming to achieve optimal milk quality in both single- and multi-breed scenarios. The tendency to move to SI is mainly related to good milk performance of SI cows coupled with their robustness, high carcass value, high market value of calves, and adaptability to mountain farming system.