Biosecurity is a set of practices applied to prevent the introduction and spread of diseases in animal farms. Mastitis is one of the most important diseases affecting dairy cows worldwide.This work aimed to evaluate the impact of biosecurity practices, environmental hygiene, and sanitation on milk quality, quantity, and prevalence of subclinical mastitis in some Egyptian dairy cattle farms. Along six months, Daily milk yield (DMY) was recorded and the total average milk production (TAMP) was calculated for six randomly selected cows of each of three farms (in Giza, Ismailia, and Alexandria Governorates, Egypt). A detailed questionnaire was also designed and filled out to detect the different biosecurity practices and hygienic levels. Milk, water, feed, and bedding samples were collected. In addition, swabs from workers’ hands, cows’ teats (before and after sanitation with Iodine 1% solution), equipment, and milking parlour surfaces were collected to assess the milk quality and environmental hygiene. Total bacterial count (TBC) and total coliform count (TCC) were determined. Subclinical mastitis (SCM) was detected using the White Side Test (WST). The obtained data revealed variable biosecurity practices in the investigated farms with different levels of environmental hygiene. A negative correlation was detected between the produced quantity and microbial quality of produced milk, besides the prevalence of SCM. Data reflected the negative impact of poor environmental hygiene on milk production and highlighted the role of following proper sanitary measures and biosecurity practices for improving milk production in dairy farms.