The objective of this study was to determinate the effect of bioclimate and milking frequency on milk production of Holstein dairy cows in summer. Measured properties were microclimate: average barn airspace temperature (BAT), relative humidity, temperature-humidity index (THI). The study lasted thirty-one days (data analyzed daily), in the period from 1 June 2013 to 1 July 2013 with BAT from 11.59 to 28.31 degrees C. Data comes from University farm. The farm is situated in aboice (GPS49 degrees 0'51.786"N, 16 degrees 36'14.809"E). Total number of cows was divided into two groups by milk yield. Almost 40 % of cows milked more than 25 kg of milk per day per cow so cows are milked twice during the morning. The other more than 60 % of dairy cows milked per day less than 25 kg of milk per day, and these cows are milked once during the morning. Based on the correlation of milk production data of Holstein cows was found that with increasing BAT and THI statistically significantly reduces average morning milk yield per cow on the second morning milking (at 8.00 a.m.) (BAT: r = -0.48, P < 0.01, THI: r = -0.47, P < 0.01) and total average morning milk yield per cow in dairy cows milked twice during the morning (BAT: r = -0.36, P < 0.05, THI: r = -0.35, P > 0.05). Furthermore, the results indicate that with increasing BAT, THI was reduced second morning total milk yield in dairy cows milked twice during the morning (at 8.00 a.m.) (BAT: r = -0.48, P < 0.01, THI: r = -0.47, P <0.01) and total milk yield in dairy cows twice milked during the morning (BAT: r = -0.37, P < 0.05, THI: r = -0.36, P < 0.05). Other analyzed parameters were not affected by microclimate (P > 0.05).