Milk production in dairy cattle is increasingly challenged by thermal variability. This underscores the need for reliable assessment of microclimatic conditions and their interaction with animal- and management-related factors to ensure sustainable dairy production. The aim of this study was to evaluate the effects of thermal variability and selected environmental and biological factors on key milk production traits in dairy cattle. The influence of fixed factors related to production conditions and microclimatic variability, including the Temperature–Humidity Index (THI), on daily milk yield (MY), milk fat content (MF), and milk protein content (MP) was assessed. The study used a dataset covering two observation periods of daily milk production traits in cows of different breeds (Simmental, Holstein-Friesian, Red Holstein, and Brown Swiss) reared in three regions of the Republic of Serbia (Mačva, Podunavlje, and Šumadija), enabling an assessment of thermal variability under diverse production and microclimatic conditions. The expression and variability of the investigated traits were determined using the PROC FREQ and PROC MEANS procedures, while the effects of individual factors were analysed using general linear and regression models, with results expressed as least squares means. All examined factors showed a highly significant effect on MY, MF, and MP (p < 0.0001). Although the overall level of heat stress was moderate, milk production was highest within the THI range of 51–60. These findings demonstrate that thermal variability significantly influences milk production and composition and highlight the importance of integrating microclimatic indicators into sustainability-oriented dairy management and breeding strategies.