The objective of the study was to determine the effect of Bacillus subtilis supplementation on the production of heat-stressed cows in a tropical environment during summer and rainy seasons. Twenty-eight lactating crossbred Holstein-Friesian cows (89 ± 50 days in milk) on four farms in Thailand were randomly assigned to treatment sequences in a replicated 4 × 4 Latin square design. Each 21 days of experimental period included 13 days of adaptation followed by data collection from day 14 through 21. A basal ration was fed to all cows. Cows were individually fed 0 (CON), 0.5 × 1011 (BS0.5), 1 × 1011 (BS1) or 2 × 1011 CFU (BS2) of B. subtilis/day. Throughout the study, microclimatic conditions inside the barns were recorded. Milk weights were measured on days 14 and 21, and milk samples were collected on day 21. Whole blood samples were collected for haematological and biochemical analyses only during the first period of the study. Milk yield increased (P < 0.01) linearly as the level of B. subtilis increased. The temperature-humidity index (THI) was negatively related to milk yield (r = −0.52, P < 0.01), protein (r = −0.51, P < 0.01) and lactose (r = −0.49, P < 0.01). Cows fed B. subtilis at the level of 2 × 1011 CFU/day produced 1.7 kg/day more milk. So, it was indicated that milk yield increased linearly with the level of supplemented B. subtilis, and the supplementation did not exert any effect on haematological and biochemical parameters.